Renilla reniformis fluorescent proteins, nucleic acids encoding the fluorescent proteins and the use thereof in diagnostics, high throughput screening and novelty items

ABSTRACT

Isolated and purified nucleic acids encoding green fluorescent proteins from  Renilla reniformis  and the green fluorescent protein encoded thereby are also provided. Mutants of the nucleic acid molecules and the modified encoded proteins are also provided. Compositions and combinations comprising the green fluorescent proteins and/or the luciferase are further provided.

RELATED APPLICATIONS

[0001] Benefit of priority under 35 U.S.C. §119(e) is claimed to U.S.provisional application Serial No. 60/189,691, filed Mar. 15, 2000, toBryan et al., entitled “RENILLA RENIFORMIS FLUORESCENT PROTEINS, NUCLEICACIDS ENCODING THE FLUORESCENT PROTEINS AND THE USE THEREOF INDIAGNOSTICS, HIGH THROUGHPUT SCREENING AND NOVELTY ITEMS” is claimed.

[0002] This application is related to allowed U.S. application Ser. No.09/277,716, filed Mar. 26, 1999, to Bruce Bryan and ChristopherSzent-Gyorgyi, entitled “LUCIFERASES, FLUORESCENT PROTEINS, NUCLEICACIDS ENCODING THE LUCIFERASES AND FLUORESCENT PROTEINS AND THE USETHEREOF IN DIAGNOSTICS, HIGH THROUGHPUT SCREENING AND NOVELTY ITEMS.”This application is related to International PCT application No. WO99/49019 to Bruce Bryan and Prolume, LTD., entitled “LUCIFERASES,FLUORESCENT PROTEINS, NUCLEIC ACIDS ENCODING THE LUCIFERASES ANDFLUORESCENT PROTEINS AND THE USE THEREOF IN DIAGNOSTICS, HIGH THROUGHPUTSCREENING AND NOVELTY ITEMS.”

[0003] This application is also related to subject matter in U.S.application Ser. No. 08/757,046, filed Nov. 25, 1996, to Bruce Bryanentitled “BIOLUMINESCENT NOVELTY ITEMS”, now U.S. Pat. No. 5,876,995,issued Mar. 2, 1999, and in U.S. application Ser. No. 08/597,274, filedFeb. 6, 1996, to Bruce Bryan, entitled “BIOLUMINESCENT NOVELTY ITEMS”.This application is also related to U.S. application Ser. No.08/908,909, filed Aug. 8, 1997, to Bruce Bryan entitled “DETECTION ANDVISUALIZATION OF NEOPLASTIC TISSUE AND OTHER TISSUES”. The applicationis also related to U.S. application Ser. No. 08/990,103, filed Dec. 12,1997, to Bruce Bryan entitled “APPARATUS AND METHODS FOR DETECTING ANDIDENTIFYING INFECTIOUS AGENTS”.

[0004] Where permitted, the subject matter of each of the above notedapplications and patents is herein incorporated by reference in itsentirety.

FIELD OF INVENTION

[0005] Provided herein are isolated and purified nucleic acids andencoded fluorescent proteins from Renilla reniformis and uses thereof.

BACKGROUND OF THE INVENTION

[0006] Luminescence is a phenomenon in which energy is specificallychanneled to a molecule to produce an excited state. Return to a lowerenergy state is accompanied release of a photon (hy). Luminescenceincludes fluorescence, phosphorescence, chemiluminescence andbioluminescence. Bioluminescence is the process by which livingorganisms emit light that is visible to other organisms. Luminescencemay be represented as follows:

A+B→X*+Y

X*→X+hv,

[0007] where X* is an electronically excited molecule and hy representslight emission upon return of X* to a lower energy state. Where theluminescence is bioluminescence, creation of the excited state derivesfrom an enzyme catalyzed reaction. The color of the emitted light in abioluminescent (or chemiluminescent or other luminescent) reaction ischaracteristic of the excited molecule, and is independent from itssource of excitation and temperature.

[0008] An essential condition for bioluminescence is the use ofmolecular oxygen, either bound or free in the presence of a luciferase.Luciferases, are oxygenases, that act on a substrate, luciferin, in thepresence of molecular oxygen and transform the substrate to an excitedstate. Upon return to a lower energy level, energy is released in theform of light (for reviews see, e.g., McElroy et al. (1966) in MolecularArchitecture in Cell Physiology, Hayashi et al., eds., Prentice-Hall,Inc., Englewood Cliffs, N.J., pp. 63-80; Ward et al., Chapter 7 inChemi-and Bioluminescence, Burr, ed., Marcel Dekker, Inc. NY,pp.321-358; Hastings, J. W. in (1995) Cell Physiology Source Book, N.Sperelakis (ed.), Academic Press, pp 665-681; Luminescence, Narcosis andLife in the Deep Sea, Johnson, Vantage Press, NY, see, esp. pp. 50-56).

[0009] Though rare overall, bioluminescence is more common in marineorganisms than in terrestrial organisms. Bioluminescence has developedfrom as many as thirty evolutionarily distinct origins and, thus, ismanifested in a variety of ways so that the biochemical andphysiological mechanisms responsible for bioluminescence in differentorganisms are distinct. Bioluminescent species span many genera andinclude microscopic organisms, such as bacteria (primarily marinebacteria including Vibrio species), fungi, algae and dinoflagellates, tomarine organisms, including arthropods, mollusks, echinoderms, andchordates, and terrestrial organism including annelid worms and insects.

[0010] Assays Employing Bioluminescence

[0011] During the past twenty years, high-sensitivity biochemical assaysused in research and in medicine have increasingly employed luminescenceand fluorescence rather than radioisotopes. This change has been drivenpartly by the increasing expense of radioisotope disposal and partly bythe need to find more rapid and convenient assay methods. More recently,the need to perform biochemical assays in situ in living cells and wholeanimals has driven researchers toward protein-based luminescence andfluorescence. The uses of firefly luciferase for ATP assays, aequorinand obelin as calcium reporters, Vargula luciferase as aneurophysiological indicator, and the Aequorea green fluorescent proteinas a protein tracer and pH indicator show the potential ofbioluminescence-based methods in research laboratories.

[0012] Bioluminescence is also beginning to directly impact medicine andbiotechnology; for example, Aequorea green fluorescent protein (GFP) isemployed to mark cells in murine model systems and as a reporter in highthroughput drug screening. Renilla luciferase is under development foruse in diagnostic platforms.

[0013] Bioluminescence Generating Systems

[0014] Bioluminescence, as well as other types of chemiluminescence, isused for quantitative determinations of specific substances in biologyand medicine. For example, luciferase genes have been cloned andexploited as reporter genes in numerous assays, for many purposes. Sincethe different luciferase systems have different specific requirements,they may be used to detect and quantify a variety of substances. Themajority of commercial bioluminescence applications are based on firefly(Photinus pyralis) luciferase. One of the first and still widely usedassays involves the use of firefly luciferase to detect the presence ofATP. It is also used to detect and quantify other substrates orco-factors in the reaction. Any reaction that produces or utilizesNAD(H), NADP(H) or long chain aldehyde, either directly or indirectly,can be coupled to the light-emitting reaction of bacterial luciferase.

[0015] Another luciferase system that has been used commercially foranalytical purposes is the Aequorin system. The purified jellyfishphotoprotein, aequorin, is used to detect and quantify intracellularCa²⁺ and its changes under various experimental conditions. The Aequorinphotoprotein is relatively small (˜20 kDa), nontoxic, and can beinjected into cells in quantities adequate to detect calcium over alarge concentration range (3×10⁻⁷ to 10⁻⁴ M).

[0016] Because of their analytical utility, luciferases and substrateshave been studied and well-characterized and are commercially available(e.g., firefly luciferase is available from Sigma, St. Louis, Mo., andBoehringer Mannheim Biochemicals, Indianapolis, Ind.; recombinantlyproduced firefly luciferase and other reagents based on this gene or foruse with this protein are available from Promega Corporation, Madison,Wis.; the aequorin photoprotein luciferase from jellyfish and luciferasefrom Renilla are commercially available from Sealite Sciences, Bogart,Ga.; coelenterazine, the naturally-occurring substrate for theseluciferases, is available from Molecular Probes, Eugene, Oreg.). Theseluciferases and related reagents are used as reagents for diagnostics,quality control, environmental testing and other such analyses.

[0017] Because of the utility of luciferases as reagents in analyticalsystems and the potential for use in high throughput screening systems,there is a need to identify and isolated a variety of luciferases thathave improved or different spectral properties compared to thosepresently available. For all these reasons, it would be advantageous tohave luciferases from a variety of species, such as Gaussia and variousRenilla species available.

[0018] Fluorescent Proteins

[0019] Reporter genes, when co-transfected into recipient cells with agene of interest, provide a means to detect transfection and otherevents. Among reporter genes are those that encode fluorescent proteins.The bioluminescence generating systems described herein are among thoseused as reporter genes. To increase the sensitivity bioluminescencegenerating systems have been combined with fluorescent compounds andproteins, such as naturally fluorescent phycobiliproteins. Also ofinterest are the fluorescent proteins that are present in a variety ofmarine invertebrates, such as the green and blue fluorescent proteins,particularly the green fluorescent protein (GFP) of Aequorea victoria.

[0020] The green fluorescent proteins (GFP) constitute a class ofchromoproteins found only among certain bioluminescent coelenterates.These accessory proteins are fluorescent and function as the ultimatebioluminescence emitter in these organisms by accepting energy fromenzyme-bound, excited-state oxyluciferin (e.g., see Ward et al. (1979)J. Biol. Chem. 254:781-788; Ward et al. (1978) Photochem. Photobiol.27:389-396; Ward et al. (1982) Biochemistry 21:4535-4540).

[0021] The best characterized GFPs are those isolated from the jellyfishspecies Aequorea, particularly Aequorea victoria (A. victoria) andAequorea forskålea (Ward et al. (1982) Biochemistry 21:4535-4540;Prendergast et al. (1978) Biochemistry 17:3448-3453). Purified A.victoria GFP is a monomeric protein of about 27 Kda that absorbs bluelight with excitation wavelength maximum of 395 nm, with a minor peak at470 nm, and emits green fluorescence with an emission wavelength ofabout 510 nm and a minor peak near 540 nm (Ward et al. (1979) Photochem.Photobiol. Rev 4:1-57). This GFP has certain limitations. The excitationmaximum of the wildtype GFP is not within the range of wavelengths ofstandard fluorescein detection optics.

[0022] The detection of green fluorescence does not require anyexogenous substrates or co-factors. Instead, the high level offluorescence results from the intrinsic chromophore of the protein. Thechromophore includes modified amino acid residues within the polypeptidechain. For example, fluorescent chromophore of A. victoria GFP isencoded by the hexapeptide sequence, FSYGVQ, encompassing amino acidresidues 64-69. The chromophore is formed by the intramolecularcyclization of the polypeptide backbone at residues Ser65 and Gly67 andthe oxidation of the α-β bond of residue Tyr66 (e.g., see Cody et al.(1993) Biochemistry 32:1212-1218; Shimomura (1978) FEBS Letters104:220-222; Ward et al. (1989) Photochem. Photobiol. 49:62S). Theemission spectrum of the isolated chromophore and the denatured proteinat neutral Ph do not match the spectrum of the native protein,suggesting that chromophore formation occurs post-translationally (e.g.,see Cody et al. (1993) Biochemistry 32:1212-1218).

[0023] In addition, the crystal structure of purified A. victoria GFPhas been determined (e.g., see Ormö (1996) Science 273:1392-1395). Thepredominant structural features of the protein are an 11-stranded βbarrel that forms a nearly perfect cylinder wrapping around a singlecentral α-helix, which contains the modifiedp-hydroxybenzylideneimadaxolidinone chromophore. The chromophore iscentrally located within the barrel structure and is completely shieldedfrom exposure to bulk solvent.

[0024] DNA encoding an isotype of A. victoria GFP has been isolated andits nucleotide sequence has been determined (e.g., see Prasher (1992)Gene 111:229-233). The A. victoria CDNA contains a 714 nucleotide openreading frame that encodes a 238 amino acid polypeptide of a calculatedM_(r) of 26,888 Da. Recombinantly expressed A. victoria GFPs retaintheir ability to fluoresce in vivo in a wide variety organisms,including bacteria (e.g., see Chalfie et al. (1994) Science 263:802-805;Miller et al. (1997) Gene 191:149-153), yeast and fungi (Fey et al.(1995) Gene 165:127-130; Straight et al. (1996) Curr. Biol. 6:1599-1608;Cormack et al. (1997) Microbiology 143:303-311), Drosophila (e.g., seeWang et al. (1994) Nature 369:400-403; Plautz (1996) Gene 173:83-87),plants (Heinlein et al. (1995); Casper et al. (1996) Gene 173:69-73),fish (Amsterdam et al. (1995)), and mammals (Ikawa et al. (1995).Aequorea GFP vectors and isolated Aequorea GFP proteins have been usedas markers for measuring gene expression, cell migration andlocalization, microtubule formation and assembly of functional ionchannels (e.g., see Terry et al. (1995) Biochem. Biophys. Res. Commun.217:21-27; Kain et al. (1995) Biotechniques 19:650-655). The A. victoriaGFP, however, is not ideal for use in analytical and diagnosticprocesses. Consequently GFP mutants have been selected with the hope ofidentifying mutants that have single excitation spectral peaks shiftedto the red.

[0025] In fact a stated purpose in constructing such mutants has been toattempt to make the A. victoria GFP more like the GFP from Renilla, butwhich has properties that make it far more ideal for use as ananalytical tool. For many practical applications, the spectrum ofRenilla GFP is be preferable to that of the Aequorea GFP, becausewavelength discrimination between different fluorophores and detectionof resonance energy transfer are easier if the component spectra aretall and narrow rather than low and broad (see, U.S. Pat. No.5,625,048). Furthermore, the longer wavelength excitation peak (475 nm)of Renilla GFP is almost ideal for fluorescein filter sets and isresistant to photobleaching, but has lower amplitude than the shorterwavelength peak at 395 nm, which is more susceptible to photobleaching(Chalfie et al. (1994) Science 263:802-805).

[0026] There exists a phylogenetically diverse and largely unexploredrepertoire of bioluminescent proteins that are a reservoir for futuredevelopment. For these reasons, it would be desirable to have a varietyof new luciferases and fluorescent proteins, particularly, Renillareniformis GFP available rather than use muteins of A. victoria GFP.Published International PCT application No. WO 99/49019 (see, also,allowed U.S. application Ser. No. 09/277,716) provides a variety of GFPsincluding those from Renilla species. It remains desirable to have avariety of GFPs and luciferases available in order to optimize systemsfor particular applications and to improve upon existing methods.Therefore, it is an object herein to provide isolated nucleic acidmolecules encoding Renilla reniformis GFP and the protein encodedthereby. It is also an object herein to provide bioluminescencegenerating systems that include the luciferases, luciferins, and alsoinclude Renilla reniformis GFP.

SUMMARY OF THE INVENTION

[0027] Isolated nucleic acid molecules that encode Renilla reniformisfluorescent proteins are provided. Nucleic acid probes and primersderived therefrom are also provided. Functionally equivalent nucleicacids, such as those that hybridize under conditions of high stringencyto the disclosed molecules and those that have high sequence identity,are also contemplated. Nucleic acid molecules and the encoded proteinsare set forth in SEQ ID Nos. 23-27, an exemplary mutein is set forth inSEQ ID No. 33. Also contemplated are nucleic acid molecules that encodethe protein set forth in SEQ ID No. 27.

[0028] Host cells, including bacterial, yeast and mammalian host cells,and plasmids for expression of the nucleic acids encoding the Renillareniformis green fluorescent protein (GFP), are also provided.Combinations of luciferases and the Renilla reniformis GFP are alsoprovided.

[0029] The genes can be modified by substitution of codons optimized forexpression in selected host cells or hosts, such as humans and othermammals, or can be mutagenized to alter the emission properties.Mutations that alter spectral properties are also contemplated.

[0030] Such mutations may be identified by substituting each codon withone encoding another amino acid, such as alanine, and determining theeffect on the spectral properties of the resulting protein. Particularregions of interest are those in which corresponding the sites mutatedin other GFPs, such Aequora to produce proteins with altered spectralproperties are altered.

[0031] The Renilla reniformis GFP may be used in combination withnucleic acids encoding luciferases, such as those known to those ofskill in the art and those that are described in copending allowed U.S.application Ser. No. 09/277,716 (see, also, Published International PCTapplication No. WO 99/49019).

[0032] Compositions containing the Renilla reniformis GFP or the Renillareniformis GFP and luciferase combination are provided. The compositionscan take any of a number of forms, depending on the intended method ofuse therefor. In certain embodiments, for example, the compositionscontain a Gaussia luciferase, Gaussia luciferase peptide or Gaussialuciferase fusion protein, formulated for use in luminescent noveltyitems, immunoassays, donors in FET (fluorescent energy transfer) assays,FRET (fluorescent resonance energy transfer) assays, HTRF (homogeneoustime-resolved fluorescence) assays or used in conjunction withmulti-well assay devices containing integrated photodetectors, such asthose described herein.

[0033] The bioluminescence-generating system includes, in addition tothe luciferase a Renilla reniformis GFP or mutated form thereof. Thesecompositions can be used in a variety of methods and systems, such asincluded in conjunction with diagnostic systems for the in vivodetection of neoplastic tissues and other tissues, such as those methodsdescribed herein.

[0034] Combinations of the Renilla reniformis GFP with an articles ofmanufacture to produce novelty items are provided. These novelty itemsare designed for entertainment, recreation and amusement, and include,but are not limited to: toys, particularly squirt guns, toy cigarettes,toy “Halloween” eggs, footbags and board/card games; finger paints andother paints, slimy play material; textiles, particularly clothing, suchas shirts, hats and sports gear suits, threads and yarns; bubbles inbubble making toys and other toys that produce bubbles; balloons;figurines; personal items, such as cosmetics, bath powders, bodylotions, gels, powders and creams, nail polishes, make-up, toothpastesand other dentifrices, soaps, body paints, and bubble bath; items suchas inks, paper; foods, such as gelatins, icings and frostings; fish foodcontaining luciferins and transgenic fish, particularly transgenic fishthat express a luciferase; plant food containing a luciferin orluciferase, preferably a luciferin for use with transgenic plants thatexpress luciferase; and beverages, such as beer, wine, champagne, softdrinks, and ice cubes and ice in other configurations; fountains,including liquid “fireworks” and other such jets or sprays or aerosolsof compositions that are solutions, mixtures, suspensions, powders,pastes, particles or other suitable form. The combinations optionallyinclude a bioluminescence generating system. The bioluminescencegenerating systems can be provided as two compositions: a firstcomposition containing a luciferase and a second composition containingone or more additional components of a bioluminescence generatingsystem.

[0035] Any article of manufacture that can be combined with abioluminescence-generating system as provided herein and thereby provideentertainment, recreation and/or amusement, including use of the itemsfor recreation or to attract attention, such as for advertising goodsand/or services that are associated with a logo or trademark iscontemplated herein. Such uses may be in addition to or in conjunctionwith or in place of the ordinary or normal use of such items. As aresult of the combination, the items glow or produce, such as in thecase of squirt guns and fountains, a glowing fluid or spray of liquid orparticles. The novelty in the novelty item derives from itsbioluminescence.

[0036] GFPS

[0037] Isolated nucleic acids that encode GFP from Renilla reniformisare provided herein. Also provided are isolated and purified nucleicacids that encode a component of the bioluminescence generating systemand a the green fluorescent protein (GFP) (see SEQ ID Nos. 23-27). Inparticular, nucleic acid molecules that encode Renilla reniformis greenfluorescent protein (GFPs) and nucleic acid probes and primers derivedtherefrom are provided. Nucleic acid molecules encoding Renillareniformis GFP are provided (see SEQ ID Nos. 23-26).

[0038] Nucleic acid probes and primers containing 14, 16, 30, 100 ormore contiguous nucleotides from any of SEQ ID Nos. 23-26. Nucleic acidprobes can be labeled, which if needed, for detection, containing atleast about 14, preferably at least about 16, or, if desired, 20 or 30or more, contiguous nucleotides of sequence of nucleotides encoding theRenilla reniformis GFP.

[0039] Methods using the probes for the isolation and cloning ofGFP-encoding DNA in Renilla reniformis are also provided. Vectorscontaining DNA encoding the Renilla reniformis GFP are provided. Inparticular, expression vectors that contain DNA encoding a Renillareniformis or in operational association with a promoter element thatallows for the constitutive or inducible expression of Renillareniformis.

[0040] The vectors are capable of expressing the Renilla reniformis GFPin a wide variety of host cells. Vectors for producing chimeric Renillareniformis GFP/luciferase fusion proteins and/or polycistronic mRNAcontaining a promoter element and a multiple cloning site locatedupstream or downstream of DNA encoding Renilla reniformis GFP are alsoprovided.

[0041] Recombinant cells containing heterologous nucleic acid encoding aRenilla reniformis GFP are also provided. Purified Renilla reniformisGFP peptides and compositions containing the Renilla GFPs and GFPpeptides alone or in combination with at least one component of abioluminescence-generating system, such as a Renilla luciferase, areprovided. The Renilla GFP and GFP peptide compositions can be used, forexample, to provide fluorescent illumination of novelty items or used inmethods of detecting and visualizing neoplastic tissue and othertissues, detecting infectious agents using immunoassays, such homogenousimmunoassays and in vitro fluorescent-based screening assays usingmulti-well assay devices, or provided in kits for carrying out any ofthe above-described methods. In particular, these proteins may be usedin FP (fluorescence polarization) assays, FET (fluorescent energytransfer) assays, FRET (fluorescent resonance energy transfer) assaysand HTRF (homogeneous time-resolved fluorescence) assays and also in theBRET assays and sensors provided herein.

[0042] Non-radioactive energy transfer reactions, such as FET or FRET,FP and HTRF assays, are homogeneous luminescence assays based on energytransfer are carried out between a donor luminescent label and anacceptor label (see, e.g., Cardullo et al. (1988) Proc. Natl. Acad. Sci.U.S.A. 85:8790-8794; Peerce et al. (1986) Proc. Natl. Acad. Sci. U.S.A.83:8092-8096; U.S. Pat. No. 4,777,128; U.S. Pat. No. 5,162,508; U.S.Pat. No. 4,927,923; U.S. Pat. No. 5,279,943; and International PCTApplication No. WO 92/01225). Non-radioactive energy transfer reactionsusing GFPs have been developed (see, International PCT application Nos.WO 98/02571 and WO 97/28261). Non-radioactive energy transfer reactionsusing GFPs and luciferases, such as a luciferase and its cognate GFP (ormultimers thereof), such as in a fusion protein, are contemplatedherein.

[0043] Nucleic acids that exhibit substantial sequence identity with thenucleic acids provided herein are also contemplated. These are nucleicacids that can be produced by substituting codons that encodeconservative amino acids and also nucleic acids that exhibit at leastabout 80%, preferably 90 or 95% sequence identity. Sequence identityrefers to identity as determined using standard programs with defaultgap penalties and other defaults as provided by the manufacturerthereof.

[0044] The nucleic acids provide an opportunity to produce luciferasesand GFPs, which have advantageous application in all areas in whichluciferase/luciferins and GFPs have application. The nucleic acids canbe used to obtain and produce GFPs and GFPs from other, particularlyRenilla species using the probes described herein that correspond toconserved regions. These GFPs have advantageous application in all areasin which GFPs and/or luciferase/luciferins have application. Forexample, The GFP's provide a means to amplify the output signal ofbioluminescence generating systems. Renilla GFP has a single excitationabsorbance peak in blue light (and around 498 nm) and a predominantlysingle emission peak around 510 nm (with a small shoulder near 540).This spectra provides a means for it to absorb blue light andefficiently convert it to green light. This results in an amplificationof the output. When used in conjunction with a bioluminescencegenerating system that yields blue light, such as Aequorea or Renilla orVargula (Cypridina), the output signal for any application, includingdiagnostic applications, is amplified. In addition, this green light canserve as an energy donor in fluorescence-based assays, such asfluorescence polarization assays, FET (fluorescent energy transfer)assays, FRET (fluorescent resonance energy transfer) assays and HTRF(homogeneous time-resolved fluorescence) assays. Particular assays,herein referred to as BRET (bioluminescence resonance energy transferassays in which energy is transferred from a bioluminescence reaction ofa luciferase to a fluorescent protein), are provided.

[0045] Non-radioactive energy transfer reactions, such as FET or FRET,FP and HTRF assays, are homogeneous luminescence assays based on energytransfer that are carried out between a donor luminescent label and anacceptor label (see, e.g., Cardullo et al. (1988) Proc. Natl. Acad. Sci.U.S.A. 85:8790-8794; Peerce et al. (1986) Proc. Natl. Acad. Sc. U.S.A.83:8092-8096; U.S. Pat. No. 4,777,128; U.S. Pat. No. 5,162,508; U.S.Pat. No. 4,927,923; U.S. Pat. No. 5,279,943; and International PCTApplication No. WO 92/01225). Non-radioactive energy transfer reactionsusing GFPs have been developed (see, International PCT application Nos.WO 98/02571 and WO 97/28261).

[0046] Mutagenesis of the GFPs is contemplated herein, particularlymutagenesis that results in modified GFPs that have red-shiftedexcitation and emission spectra. The resulting systems have higheroutput compared to the unmutagenized forms. These GFPs may be selectedby random mutagenesis and selection for GFPs with altered spectra or byselected mutagenesis of the chromophore region of the GFP.

[0047] The DNA may be introduced as a linear DNA molecule (fragment) ormay be included in an expression vector for stable or transientexpression of the encoding DNA. In certain embodiments, the cellscontain DNA or RNA encoding a Renilla GFP also express the recombinantRenilla GFP or polypeptide. It is preferred that the cells are selectedto express functional GFPs that retain the ability to fluorescence andthat are not toxic to the host cell. In some embodiments, cells may alsoinclude heterologous nucleic acid encoding a component of abioluminescence-generating system, preferably a photoprotein orluciferase. In preferred embodiments, the nucleic acid encoding thebioluminescence-generating system component is isolated from the speciesAequorea, Vargula, Pleuromamma, Ptilosarcus or Renilla. In morepreferred embodiments, the bioluminescence-generating system componentis a Renilla reniformis luciferase or mulleri including the amino acidsequence set forth in SEQ ID No. 18 or the Pleuromamma luciferase setforth in SEQ ID No. 28, or the Gaussia luciferase set forth in SEQ IDNo. 19.

[0048] The GFPs provided herein may be used in combination with anysuitable bioluminescence generating system, but is preferably used incombination with a Renilla or Aequorea, Pleuromamma or Gaussialuciferase.

[0049] Purified Renilla GFPs, particularly purified Renilla reniformisGFP peptides are provided. Presently preferred Renilla GFP for use inthe compositions herein is Renilla reniformis GFP including the sequenceof amino acids set forth above and in the Sequence Listing.

[0050] Fusions of the nucleic acid, particularly DNA, encoding RenillaGFP with DNA encoding a luciferase are also provided herein.

[0051] The cells that express functional luciferase and/or GFP, whichmay be used alone or in conjunction with a bioluminescence-generatingsystem, in cell-based assays and screening methods, such as thosedescribed herein.

[0052] Presently preferred host cells for expressing GFP and luciferaseare bacteria, yeasts, fungi, plant cells, insect cells and animal cells.

[0053] The luciferases and GFPs or cells that express them also may beused in methods of screening for bacterial contamination and methods ofscreening for metal contaminants. To screen for bacterial contamination,bacterial cells that express the luciferase and/or GFP are put inautoclaves or in other areas in which testing is contemplated. Aftertreatment or use of the area, the area is tested for the presence ofglowing bacteria. Presence of such bacteria is indicative of a failureto eradicate other bacteria. Screening for heavy metals and otherenvironmental contaminants can also be performed with cells that containthe nucleic dependent upon the particular heavy metal or contaminant.

[0054] The systems and cells provided herein can be used for highthroughout screening protocols, intracellular assays, medical diagnosticassays, environmental testing, such as tracing bacteria in watersupplies, in conjunction with enzymes for detecting heavy metals, inspores for testing autoclaves in hospital, foods and industrialautoclaves. Non-pathogenic bacteria containing the systems can beincluded in feed to animals to detect bacterial contamination in animalproducts and in meats.

[0055] Compositions containing a Renilla GFP are provided. Thecompositions can take any of a number of forms, depending on theintended method of use therefor. In certain embodiments, for example,the compositions contain a Renilla GFP or GFP peptide, preferablyRenilla mulleri GFP or Renilla reniformis GFP peptide, formulated foruse in luminescent novelty items, immunoassays, FET (fluorescent energytransfer) assays, FRET (fluorescent resonance energy transfer) assays,HTRF (homogeneous time-resolved fluorescence) assays or used inconjunction with multi-well assay devices containing integratedphotodetectors, such as those described herein. In other instances, theGFPs are used in beverages, foods or cosmetics.

[0056] Compositions that contain a Renilla reniformis GFP or GFP peptideand at least one component of a bioluminescence-generating system,preferably a luciferase, luciferin or a luciferase and a luciferin, areprovided. In preferred embodiments, the luciferase/luciferinbioluminescence-generating system is selected from those isolated from:an insect system, a coelenterate system, a ctenophore system, abacterial system, a mollusk system, a crustacea system, a fish system,an annelid system, and an earthworm system. Bioluminescence-generatingsystems include those isolated from Renilla, Aequorea, and Vargula,Gaussia and Pleuromamma.

[0057] Combinations containing a first composition containing a Renillareniformis GFP or Ptilosarcus GFP or mixtures thereof and a secondcomposition containing a bioluminescence-generating system for use withinanimate articles of manufacture to produce novelty items are provided.These novelty items, which are articles of manufacture, are designed forentertainment, recreation and amusement, and include, but are notlimited to: toys, particularly squirt guns, toy cigarettes, toy“Halloween” eggs, footbags and board/card games; finger paints and otherpaints, slimy play material; textiles, particularly clothing, such asshirts, hats and sports gear suits, threads and yarns; bubbles in bubblemaking toys and other toys that produce bubbles; balloons; figurines;personal items, such as bath powders, body lotions, gels, powders andcreams, nail polishes, cosmetic including make-up, toothpastes and otherdentifrices, soaps, cosmetics, body paints, and bubble bath, bubblesmade from non-detergent sources, particularly proteins such as albuminand other non-toxic proteins; in fishing lures and glowing transgenicworms, particularly crosslinked polyacrylamide containing a fluorescentprotein and/or components of a bioluminescence generating system, whichglow upon contact with water; items such as inks, paper; foods, such asgelatins, icings and frostings; fish food containing luciferins andtransgenic animals, such as transgenic fish, worms, monkeys, rodents,ungulates, ovine, ruminants and others, that express a luciferase and/orRenilla reniformis GFP; transgenic worms that express Renilla reniformisGFP and are used as lures; plant food containing a luciferin orluciferase, preferably a luciferin for use with transgenic plants thatexpress luciferase and Renilla reniformis GFP, transgenic plants thatexpress Renilla reniformis GFP, particularly ornamental plants, such asorchids, roses, and other plants with decorative flowers; transgenicplants and animals in which the Renilla reniformis GFP is a marker fortracking introduction of other genes; and beverages, such as beer, wine,champagne, soft drinks, milk and ice cubes and ice in otherconfigurations containing Renilla reniformis GFP; fountains, includingliquid “fireworks” and other such jets or sprays or aerosols ofcompositions that are solutions, mixtures, suspensions, powders, pastes,particles or other suitable form.

[0058] Any article of manufacture that can be combined with abioluminescence-generating system and Renilla reniformis GFP or withjust a Renilla reniformis GFP, as provided herein, that thereby provideentertainment, recreation and/or amusement, including use of the itemsfor recreation or to attract attention, such as for advertising goodsand/or services that are associated with a logo or trademark iscontemplated herein. Such uses may be in addition to or in conjunctionwith or in place of the ordinary or normal use of such items. As aresult of the combination, the items glow or produce, such as in thecase of squirt guns and fountains, a glowing fluid or spray of liquid orparticles.

[0059] Methods for diagnosis and visualization of tissues in vivo or insitu using compositions containing a Renilla reniformis GFP and/or aRenilla reniformis or mulleri luciferase or others of the luciferasesand/or GFPs provided herein are provided. For example, the Renillareniformis GFP protein can be used in conjunction with diagnosticsystems that rely on bioluminescence for visualizing tissues in situ.The systems are particularly useful for visualizing and detectingneoplastic tissue and specialty tissue, such as during non-invasive andinvasive procedures. The systems include compositions containingconjugates that include a tissue specific, particularly atumor-specific, targeting agent linked to a targeted agent, a Renillareniformis GFP, a luciferase or luciferin. The systems also include asecond composition that contains the remaining components of abioluminescence generating reaction and/or the Renilla reniformis GFP.In some embodiments, all components, except for activators, which areprovided in situ or are present in the body or tissue, are included in asingle composition.

[0060] Methods for diagnosis and visualization of tissues in vivo or insitu using compositions containing a Gaussia luciferase are provided.For example, the Gaussia luciferase or Gaussia luciferase peptide can beused in conjunction with diagnostic systems that rely on bioluminescencefor visualizing tissues in situ. The systems are particularly useful forvisualizing and detecting neoplastic tissue and specialty tissue, suchas during non-invasive and invasive procedures. The systems includecompositions containing conjugates that include a tissue specific,particularly a tumor-specific, targeting agent linked to a targetedagent, a Gaussia luciferase, a GFP or luciferin. The systems alsoinclude a second composition that contains the remaining components of abioluminescence generating reaction and/or the Gaussia luciferase. Insome embodiments, all components, except for activators, which areprovided in situ or are present in the body or tissue, are included in asingle composition.

[0061] In particular, the diagnostic systems include two compositions. Afirst composition that contains conjugates that, in preferredembodiments, include antibodies directed against tumor antigensconjugated to a component of the bioluminescence generating reaction, aluciferase or luciferin, preferably a luciferase are provided. Incertain embodiments, conjugates containing tumor-specific targetingagents are linked to luciferases or luciferins. In other embodiments,tumor-specific targeting agents are linked to microcarriers that arecoupled with, preferably more than one of the bioluminescence generatingcomponents, preferably more than one luciferase molecule.

[0062] The second composition contains the remaining components of abioluminescence generating system, typically the luciferin or luciferasesubstrate. In some embodiments, these components, particularly theluciferin are linked to a protein, such as a serum albumin, or otherprotein carrier. The carrier and time release formulations permitsystemically administered components to travel to the targeted tissuewithout interaction with blood cell components, such as hemoglobin thatdeactivates the luciferin or luciferase.

[0063] Methods for diagnosing diseases, particularly infectiousdiseases, using chip methodology (see, e.g., copending U.S. applicationSer. No. 08/990,103) a luciferase/luciferin bioluminescence-generatingsystem and a Renilla reniformis GFP are provided. In particular, thechip includes an integrated photodetector that detects the photonsemitted by the bioluminescence-generating system, particularly usingluciferase encoded by the nucleic acids provided herein and/or Renillareniformis GFP.

[0064] In one embodiment, the chip is made using an integrated circuitwith an array, such as an X-Y array, of photodetectors. The surface ofcircuit is treated to render it inert to conditions of the diagnosticassays for which the chip is intended, and is adapted, such as byderivatization for linking molecules, such as antibodies. A selectedantibody or panel of antibodies, such as an antibody specific for abacterial antigen, is affixed to the surface of the chip above eachphotodetector. After contacting the chip with a test sample, the chip iscontacted with a second antibody linked to a Renilla GFP, a chimericantibody-Renilla GFP fusion protein or an antibody linked to a componentof a bioluminescence generating system, such as a luciferase orluciferin, that are specific for the antigen. The remaining componentsof the bioluminescence generating reaction are added, and, if any of theantibodies linked to a component of a bioluminescence generating systemare present on the chip, light will be generated and detected by theadjacent photodetector. The photodetector is operatively linked to acomputer, which is programmed with information identifying the linkedantibodies, records the event, and thereby identifies antigens presentin the test sample.

[0065] Methods for generating chimeric GFP fusion proteins are provided.The methods include linking DNA encoding a gene of interest, or portionthereof, to DNA encoding a GFP coding region in the same translationalreading frame. The encoded-protein of interest may be linked in-frame tothe amino- or carboxyl-terminus of the GFP. The DNA encoding thechimeric protein is then linked in operable association with a promoterelement of a suitable expression vector. Alternatively, the promoterelement can be obtained directly from the targeted gene of interest andthe promoter-containing fragment linked upstream of the GFP codingsequence to produce chimeric GFP proteins or two produce polycistronicmRNAs that encode the Renilla reniformis GFP and a luciferase,preferably a Renilla luciferase, more preferably Renilla reniformisluciferase.

[0066] Methods for identifying compounds using recombinant cells thatexpress heterologous DNA encoding a Renilla reniformis GFP under thecontrol of a promoter element of a gene of interest are provided. Therecombinant cells can be used to identify compounds or ligands thatmodulate the level of transcription from the promoter of interest bymeasuring Renilla reniformis GFP-mediated fluorescence. Recombinantcells expressing the chimeric Renilla reniformis GFP or polycistronicmRNA encoding Renilla reniformis and a lucifierase, may also be used formonitoring gene expression or protein trafficking, or determining thecellular localization of the target protein by identifying localizedregions of GFP-mediated fluorescence within the recombinant cell.

[0067] Other assays using the GFPs and/or luciferases are contemplatedherein. Any assay or diagnostic method known used by those of skill inthe art that employ Aequora GFPs and/or other luciferases arecontemplated herein.

[0068] Kits containing the GFPs for use in the methods, including thosedescribed herein, are provided. In one embodiment, the kits containingan article of manufacture and appropriate reagents for generatingbioluminescence are provided. The kits containing such soapcompositions, with preferably a moderate Ph (between 5 and 8) andbioluminescence generating reagents, including luciferase and luciferinand the GFP are provided herein. These kits, for example, can be usedwith a bubble-blowing or producing toy. These kits can also include areloading or charging cartridge or can be used in connection with afood.

[0069] In another embodiment, the kits are used for detecting andvisualizing neoplastic tissue and other tissues and include a firstcomposition that contains the GFP and at least one component of abioluminescence generating system, and a second that contains theactivating composition, which contains the remaining components of thebioluminescence generating system and any necessary activating agents.

[0070] Thus, these kits will typically include two compositions, a firstcomposition containing the GFP formulated for systemic administration(or in some embodiments local or topical application), and a secondcomposition containing the components or remaining components of abioluminescence generating system, formulated for systemic, topical orlocal administration depending upon the application. Instructions foradministration will be included.

[0071] In other embodiments, the kits are used for detecting andidentifying diseases, particularly infectious diseases, using multi-wellassay devices and include a multi-well assay device containing aplurality of wells, each having an integrated photodetector, to which anantibody or panel of antibodies specific for one or more infectiousagents are attached, and composition containing a secondary antibody,such as an antibody specific for the infectious agent that is linked toa Renilla reniformis GFP protein, a chimeric antibody-Renillareniformis) GFP fusion protein or F(Ab)₂ antibody fragment-Renillareniformis GFP fusion protein. A second composition containing abioluminescence generating system that emits a wavelength of lightwithin the excitation range of the Renilla mulleri GFP, such as speciesof Renilla or Aequorea, for exciting the Renilla reniformis, whichproduces light that is detected by the photodetector of the device toindicate the presence of the agent.

[0072] As noted above, fusions of nucleic acid encoding the luciferasesand or GFPs provided herein with other luciferases and GFPs areprovided. Of particular interest are fusions that encode pairs ofluciferases and GFPs, such as a Renilla luciferase and a Renilla GFP (ora homodimer or other multiple of a Renilla GFP). The luciferase and GFPbind and in the presence of a luciferin will produced fluorescence thatis red shifted compared to the luciferase in the absence of the GFP.This fusion or fusions in which the GFP and luciferase are linked via atarget, such as a peptide, can be used as a tool to assess anything thatinteracts with the linker.

[0073] Muteins of the GFPs and luciferases are provided. Of particularinterest are muteins, such as temperature sensitive muteins, of the GFPand luciferases that alter their interaction, such as mutations in theRenilla luciferase and Renilla GFP that alters their interaction at acritical temperature.

[0074] Antibodies, polyclonal and monoclonal antibodies thatspecifically bind to any of the proteins encoded by the nucleic acidsprovided herein are also provided. These antibodies, monoclonal orpolyclonal, can be prepared employing standard techniques, known tothose of skill in the art. In particular, immunoglobulins or antibodiesobtained from the serum of an animal immunized with a substantially purepreparation of a luciferase or GFP provided herein or an orepitope-containing fragment thereof are provided. Monoclonal antibodiesare also provided. The immunoglobulins that are produced have, amongother properties, the ability to specifically and preferentially bind toand/or cause the immunoprecipitation of a GFP or luciferase,particularly a Renilla or Ptilosarcus GFP or a Pleuromamma, Gaussia orRenilla mulleri luciferase, that may be present in a biological sampleor a solution derived from such a biological sample.

DESCRIPTION OF THE FIGURES

[0075]FIG. 1 depicts phylogenetic relationships among the anthozoanGFPs.

[0076]FIG. 2 illustrates the underlying principle of BioluminescentResonance Energy Transfer (BRET) and its use as sensor: A) in isolation,a luciferase, preferably an anthozoan luciferase, emits blue light fromthe coelenterazine-derived chromophore; B) in isolation, a GFP,preferably an anthozoan GFP that binds to the luciferase, that isexcited with blue-green light emits green light from its integralpeptide based fluorophore; C) when the luciferase and GFP associate as acomplex in vivo or in vitro, the luciferase non-radiatively transfersits reaction energy to the GFP flurophore, which then emits the greenlight; D) any molecular interaction that disrupts the luciferase-GFPcomplex can be quantitatively monitored by observing the spectral shiftfrom green to blue light.

[0077]FIG. 3 illustrates exemplary BRET sensor architecture.

[0078]FIG. 4 depicts the substitution of altered fluorophores into thebackground of Ptilosarcus, Renilla mulleri and Renilla reniformis GFPs(the underlined regions corresponds to amino acids 56-75 of SEQ ID No.27 Renilla reniformis GFP; amino acids 59-78 of SEQ ID No. 16 Renillamulleri GFP; and amino acids 9-78 of SEQ ID No. 32 for Ptilosarcus GFP).

[0079]FIG. 5 depicts the three anthozoan fluorescent protein for which acrystal structure exists, another available commercially from Clontechas dsRed (from Discosoma striata; also known as drFP583, as in thisalignment); a dark gray background depicts amino acid conservation, anda light gray background depicts shared physicochemical properties.

[0080]FIG. 6 compares the sequences of a variety of GFPs, identifyingsites for mutation to reduce multimerization; abbreviations are asfollows: Amemonia majona is amFP486; Zoanthus sp. zFP506 and zFP538;Discosoma sp. “red” is drFP583; Clavularia sp. is cFP484; and the GFPfrom the anthozoal A. sulcata is designated FP595.

DETAILED DESCRIPTION OF THE INVENTION

[0081] A. Definitions

[0082] B. Fluorescent Proteins

[0083] 1. Green and blue fluorescent proteins

[0084] 2. Renilla reniformis GFP

[0085] C. Bioluminescence Generating Systems and Components

[0086] 1. General description

[0087] a. Luciferases

[0088] b. Luciferins

[0089] C. Activators

[0090] d. Reactions

[0091] 2. The Renilla system

[0092] 3. Ctenophore systems

[0093] 4. The aequorin system

[0094] a. Aequorin and related photoproteins

[0095] b. Luciferin

[0096] 5. Crustacean, particularly Cyrpidina systems

[0097] a. Vargula luciferase

[0098] (1) Purification from Cypridina

[0099] (2) Preparation by Recombinant Methods

[0100] b. Vargula luciferin

[0101] c. Reaction

[0102] 6. Insect bioluminescent systems including fireflies, clickbeetles, and other insect system

[0103] a. Luciferase

[0104] b. Luciferin

[0105] c. Reaction

[0106] 7. Other systems

[0107] a. Bacterial systems

[0108] (1) Luciferases

[0109] (2) Luciferins

[0110] (3) Reactions

[0111] b. Dinoflagellate bioluminescence generating systems

[0112] D. Isolation and Identification of Nucleic Acids EncodingLuciferases and GFPs

[0113] 1. Isolation of specimens of the genus Renilla

[0114] 2. Preparation of Renilla cDNA expression libraries

[0115] a. RNA isolation and cDNA synthesis

[0116] b. Construction of cDNA expression libraries

[0117] 3. Cloning of Renilla reniformis Green Fluorescent Protein

[0118] 4. Isolation and identification of DNA encoding Renilla mulleriGFP

[0119] 5. Isolation and identification of DNA encoding Renilla mulleriluciferase

[0120] E. Recombinant Expression of Proteins

[0121] 1. DNA encoding Renilla proteins

[0122] 2. DNA constructs for recombinant production of Renillareniformis and other proteins

[0123] 3. Host organisms for recombinant production of Renilla proteins

[0124] 4. Methods for recombinant production of Renilla proteins

[0125] 5. Recombinant cells expressing heterologous nucleic acidencoding luciferases and GFPs

[0126] F. Compositions and Conjugates

[0127] 1. Renilla GFP compositions

[0128] 2. Renilla luciferase compositions

[0129] 3. Conjugates

[0130] a. Linkers

[0131] b. Targeting Agents

[0132] c. Anti-tumor Antigen Antibodies

[0133] d. Preparation of the conjugates

[0134] 4. Formulation of the compositions for use in the diagnosticsystems

[0135] a. The first composition: formulation of the conjugates

[0136] b. The second composition

[0137] c. Practice of the reactions in combination with targeting agents

[0138] G. Combinations

[0139] H. Exemplary uses of Renilla reniformis GFPs and encoding nucleicacid molecules

[0140] 1. Methods for diagnosis of neoplasms and other tissues

[0141] 2. Methods of diagnosing diseases

[0142] 3. Methods for generating Renilla mulleri luciferase, Pleuromammaluciferase and Gaussia luciferase fusion proteins with Renillareniformis GFP

[0143] 4. Cell-based assays for identifying compounds

[0144] I. Kits

[0145] J. Muteins

[0146] 1. Mutation of GFP surfaces to disrupt multimerization

[0147] 2. Use of advantageous GFP surfaces with substituted fluorophores

[0148] K. Transgenic Plants and Animals

[0149] L. Bioluminescence Resonance Energy Transfer (BRET) System

[0150] 1. Design of sensors based on BRET

[0151] 2. BRET Sensor Architectures

[0152] 3. Advantages of BRET sensors

[0153] A. Definitions

[0154] Unless defined otherwise, all technical and scientific terms usedherein have the same meaning as is commonly understood by one of skillin the art to which this invention belongs. All patents, applicationsand publications of referred to throughout the disclosure areincorporated by reference in their entirety.

[0155] As used herein, chemiluminescence refers to a chemical reactionin which energy is specifically channeled to a molecule causing it tobecome electronically excited and subsequently to release a photonthereby emitting visible light. Temperature does not contribute to thischanneled energy. Thus, chemiluminescence involves the direct conversionof chemical energy to light energy.

[0156] As used herein, luminescence refers to the detectable EMradiation, generally, UV, IR or visible EM radiation that is producedwhen the excited product of an exergic chemical process reverts to itsground state with the emission of light. Chemiluminescence isluminescence that results from a chemical reaction. Bioluminescence ischemiluminescence that results from a chemical reaction using biologicalmolecules (or synthetic versions or analogs thereof) as substratesand/or enzymes.

[0157] As used herein, bioluminescence, which is a type ofchemiluminescence, refers to the emission of light by biologicalmolecules, particularly proteins. The essential condition forbioluminescence is molecular oxygen, either bound or free in thepresence of an oxygenase, a luciferase, which acts on a substrate, aluciferin. Bioluminescence is generated by an enzyme or other protein(luciferase) that is an oxygenase that acts on a substrate luciferin (abioluminescence substrate) in the presence of molecular oxygen andtransforms the substrate to an excited state, which upon return to alower energy level releases the energy in the form of light.

[0158] As used herein, the substrates and enzymes for producingbioluminescence are generically referred to as luciferin and luciferase,respectively. When reference is made to a particular species thereof,for clarity, each generic term is used with the name of the organismfrom which it derives, for example, bacterial luciferin or fireflyluciferase.

[0159] As used herein, luciferase refers to oxygenases that catalyze alight emitting reaction. For instance, bacterial luciferases catalyzethe oxidation of flavin mononucleotide (FMN) and aliphatic aldehydes,which reaction produces light. Another class of luciferases, found amongmarine arthropods, catalyzes the oxidation of Cypridina (Vargula)luciferin, and another class of luciferases catalyzes the oxidation ofColeoptera luciferin.

[0160] Thus, luciferase refers to an enzyme or photoprotein thatcatalyzes a bioluminescent reaction (a reaction that producesbioluminescence). The luciferases, such as firefly and Gaussia andRenilla luciferases, that are enzymes which act catalytically and areunchanged during the bioluminescence generating reaction. The luciferasephotoproteins, such as the aequorin photoprotein to which luciferin isnon-covalently bound, are changed, such as by release of the luciferin,during bioluminescence generating reaction. The luciferase is a proteinthat occurs naturally in an organism or a variant or mutant thereof,such as a variant produced by mutagenesis that has one or moreproperties, such as thermal stability, that differ from thenaturally-occurring protein. Luciferases and modified mutant or variantforms thereof are well known. For purposes herein, reference toluciferase refers to either the photoproteins or luciferases.

[0161] Thus, reference, for example, to “Gaussia luciferase” means anenzyme isolated from member of the genus Gaussia or an equivalentmolecule obtained from any other source, such as from another relatedcopepod, or that has been prepared synthetically. It is intended toencompass Gaussia luciferases with conservative amino acid substitutionsthat do not substantially alter activity. Suitable conservativesubstitutions of amino acids are known to those of skill in this art andmay be made generally without altering the biological activity of theresulting molecule. Those of skill in this art recognize that, ingeneral, single amino acid substitutions in non-essential regions of apolypeptide do not substantially alter biological activity (see, e.g.,Watson et al. Molecular Biology of the Gene, 4th Edition, 1987, TheBejacmin/Cummings Pub. co., p.224).

[0162] “Renilla GFP” refers to GFPs from the genus Renilla and tomutants or variants thereof. It is intended to encompass Renilla GFPswith conservative amino acid substitutions that do not substantiallyalter activity and physical properties, such as the emission spectra andability to shift the spectral output of bioluminescence generatingsystems.

[0163] Such substitutions are preferably made in accordance with thoseset forth in TABLE 1 as follows: TABLE 1 Original residue Conservativesubstitution Ala (A) Gly; Ser Arg (R) Lys Asn (N) Gln; His Cys (C) SerGln (Q) Asn Glu (E) Asp Gly (G) Ala; Pro His (H) Asn; Gln Ile (I) Leu;Val Leu (L) Ile; Val Lys (K) Arg; Gln; Glu Met (M) Leu; Tyr; Ile Phe (F)Met; Leu; Tyr Ser (S) Thr Thr (T) Ser Trp (W) Tyr Tyr (Y) Trp; Phe Val(V) Ile; Leu

[0164] Other substitutions are also permissible and may be determinedempirically or in accord with known conservative substitutions.

[0165] The luciferases and luciferin and activators thereof are referredto as bioluminescence generating reagents or components. Typically, asubset of these reagents will be provided or combined with an article ofmanufacture. Bioluminescence will be produced upon contacting thecombination with the remaining reagents. Thus, as used herein, thecomponent luciferases, luciferins, and other factors, such as O₂, Mg²⁺,Ca²⁺ are also referred to as bioluminescence generating reagents (oragents or components).

[0166] As used herein, a Renilla reniformis green fluorescent protein(GFP) refers to a fluorescent protein that is encoded by a sequence ofnucleotides that encodes the protein of SEQ ID No. 27 or to a greenfluorescent protein from Renilla reniformis having at least 80%, 90% or95% or greater sequence identity thereto; or that is encoded by asequence of nucleotides that hybridizes under high stringency along itsfull length to the coding portion of the sequence of nucleotides setforth in any of SEQ ID Nos. 23-25. A Renilla reniformis GFP is proteinthat is fluorescent and is produced in a Renilla reniformis.

[0167] As used herein, bioluminescence substrate refers to the compoundthat is oxidized in the presence of a luciferase, and any necessaryactivators, and generates light. These substrates are referred to asluciferins herein, are substrates that undergo oxidation in abioluminescence reaction. These bioluminescence substrates include anyluciferin or analog thereof or any synthetic compound with which aluciferase interacts to generate light. Preferred substrates are thosethat are oxidized in the presence of a luciferase or protein in alight-generating reaction. Bioluminescence substrates, thus, includethose compounds that those of skill in the art recognize as luciferins.Luciferins, for example, include firefly luciferin, Cypridina (alsoknown as Vargula) luciferin (coelenterazine), bacterial luciferin, aswell as synthetic analogs of these substrates or other compounds thatare oxidized in the presence of a luciferase in a reaction the producesbioluminescence.

[0168] As used herein, capable of conversion into a bioluminescencesubstrate means susceptible to chemical reaction, such as oxidation orreduction, that yields a bioluminescence substrate. For example, theluminescence producing reaction of bioluminescent bacteria involves thereduction of a flavin mononucleotide group (FMN) to reduced flavinmononucleotide (FMNH₂) by a flavin reductase enzyme. The reduced flavinmononucleotide (substrate) then reacts with oxygen (an activator) andbacterial luciferase to form an intermediate peroxy flavin thatundergoes further reaction, in the presence of a long-chain aldehyde, togenerate light. With respect to this reaction, the reduced flavin andthe long chain aldehyde are substrates.

[0169] As used herein, a bioluminescence generating system refers to theset of reagents required to conduct a bioluminescent reaction. Thus, thespecific luciferase, luciferin and other substrates, solvents and otherreagents that may be required to complete a bioluminescent reaction forma bioluminescence system. Thus a bioluminescence generating systemrefers to any set of reagents that, under appropriate reactionconditions, yield bioluminescence. Appropriate reaction conditionsrefers to the conditions necessary for a bioluminescence reaction tooccur, such as pH, salt concentrations and temperature. In general,bioluminescence systems include a bioluminescence substrate, luciferin,a luciferase, which includes enzymes luciferases and photoproteins, andone or more activators. A specific bioluminescence system may beidentified by reference to the specific organism from which theluciferase derives; for example, the Vargula (also called Cypridina)bioluminescence system (or Vargula system) includes a Vargulaluciferase, such as a luciferase isolated from the ostracod, Vargula orproduced using recombinant means or modifications of these luciferases.This system would also include the particular activators necessary tocomplete the bioluminescence reaction, such as oxygen and a substratewith which the luciferase reacts in the presence of the oxygen toproduce light.

[0170] The luciferases provided herein may be incorporated intobioluminescence generating systems and used, as appropriate, with theGFPs provided herein or with other GFPs. Similarly, the GFPs providedherein may be used with known bioluminescence generating systems.

[0171] As used herein, the amino acids, which occur in the various aminoacid sequences appearing herein, are identified according to theirwell-known, three-letter or one-letter abbreviations. The nucleotides,which occur in the various DNA molecules, are designated with thestandard single-letter designations used routinely in the art.

[0172] As used herein, a fluorescent protein refers to a protein thatpossesses the ability to fluoresce (i.e., to absorb energy at onewavelength and emit it at another wavelength). These proteins can beused as a fluorescent label or marker and in any applications in whichsuch labels would be used, such as immunoassays, CRET, FRET, and FETassays, and in the assays designated herein as BRET assays. For example,a green fluorescent protein refers to a polypeptide that has a peak inthe emission spectrum at about 510 nm.

[0173] As used herein, the term BRET (Bioluminescence Resonance EnergyTransfer) refers to non-radiative luciferase-to-FP energy transfer. Itdiffers from (Fluorescence Resonance Energy Transfer), which refers toenergy transfer between chemical fluors.

[0174] As used herein, a BRET system refers the combination of a FP, inthis case Renilla reniformis GFP and a luciferase for resonance energytransfer. BRET refers to any method in which the luciferase is used togenerate the light upon reaction with a luciferin which is thennon-radiatively transferred to a FP. The energy is transferred to a FP,particularly a GFP, which focuses and shifts the energy and emits it ata different wavelength. In preferred embodiments, the BRET systemincludes a bioluminescence generating system and a Renilla reniformisGFP. The bioluminescence generating system is preferably a Renillasystem. Hence, the preferred pair is a Renilla luciferase and a RenillaGFP, which specifically interact. Alterations in the binding will bereflected in changes in the emission spectra of light produced by theluciferase. As a result the pair can function as a sensor of externalevents.

[0175] As used herein, a biosensor (or sensor) refers to a BRET systemfor use to detect alterations in the environment in vitro or in vivo inwhich the BRET system is used.

[0176] As used herein, modulator with reference to a BRET system refersto a molecule or molecules that undergo a conformation change inresponse to interaction with another molecule thereby affecting theproximity and/or orientation of the GFP and luciferase in the BRETsystem. Modulators include, but are not limited to, a protease site, asecond messenger binding site, an ion binding molecule, a receptor, anoligomer, an enzyme substrate, a ligand, or other such binding molecule.If the GFP and luciferase are each linked to the modulator, changes inconformation alter the spacial relationship between the GFP andluciferase. The modulator can be a single entity covalently attached toone or both of the luciferase and GFP; it can be two separate entitieseach linked to either the luciferase or GFP. The modulator(s), GFP andluciferase can be a single fusion protein, or a fusion protein of atleast two of the entities. The components can be chemically linked, suchas through thiol or disulfide linkages, using linkers as providedherein. The GFP and luciferase can be linked directly or via linker,which can be a chemical linkage.

[0177] As used herein, “not strictly catalytically” means that thephotoprotein acts as a catalyst to promote the oxidation of thesubstrate, but it is changed in the reaction, since the bound substrateis oxidized and bound molecular oxygen is used in the reaction. Suchphotoproteins are regenerated by addition of the substrate and molecularoxygen under appropriate conditions known to those of skill in this art.

[0178] As used herein, “nucleic acid” refers to a polynucleotidecontaining at least two covalently linked nucleotide or nucleotideanalog subunits. A nucleic acid can be a deoxyribonucleic acid (DNA), aribonucleic acid (RNA), or an analog of DNA or RNA. Nucleotide analogsare commercially available and methods of preparing polynucleotidescontaining such nucleotide analogs are known (Lin et al. (1994) Nucl.Acids Res. 22:5220-5234; Jellinek et al. (1995) Biochemistry34:11363-11372; Pagratis et al. (1997) Nature Biotechnol. 15:68-73). Thenucleic acid can be single-stranded, double-stranded, or a mixturethereof. For purposes herein, unless specified otherwise, the nucleicacid is double-stranded, or it is apparent from the context.

[0179] As used herein, a second messenger includes, but are not limitedto, cAMP, cGMP, inositol phosphates, such as IP2 and IP3, NO (nitricoxide), Ca²⁺, ceramide; DAG and arachidonic acid.

[0180] Hence, the term “nucleic acid” refers to single-stranded and/ordouble-stranded polynucleotides, such as deoxyribonucleic acid (DNA) andribonucleic acid (RNA), as well as analogs or derivatives of either RNAor DNA. Also included in the term “nucleic acid” are analogs of nucleicacids such as peptide nucleic acid (PNA), phosphorothioate DNA, andother such analogs and derivatives.

[0181] As used herein, the term “nucleic acid molecule” and “nucleicacid fragment” are used interchangeably.

[0182] As used herein, DNA is meant to include all types and sizes ofDNA molecules including cDNA, plasmids and DNA including modifiednucleotides and nucleotide analogs.

[0183] As used herein, nucleotides include nucleoside mono-, di-, andtriphosphates. Nucleotides also include modified nucleotides, such as,but are not limited to, phosphorothioate nucleotides and deazapurinenucleotides and other nucleotide analogs.

[0184] As used herein, a nucleic acid probe is single-stranded DNA orRNA that has a sequence of nucleotides that includes at least 14contiguous bases, preferably at least 16 contiguous bases, typicallyabout 30, that are the same as (or the complement of) any 14 or morecontiguous bases set forth in any of SEQ ID No. and herein. Among thepreferred regions from which to construct probes include 5′ and/or 3′coding sequences, sequences predicted to encode regions that areconserved among Renilla species. Probes from regions conserved amongRenilla species GFPs are for isolating GFP-encoding nucleic acid fromRenilla libraries.

[0185] In preferred embodiments, the nucleic acid probes are degenerateprobes of at least 14 nucleotides, preferably 16 to 30 nucleotides, areprovided.

[0186] In preferred embodiments, the nucleic acid probes are degenerateprobes of at least 14 nucleotides, preferably 16 to 30 nucleotides, thatare based on amino acids of Renilla reniformis set forth in above.

[0187] As used herein, vector (or plasmid) refers to discrete elementsthat are used to introduce heterologous DNA into cells for eitherexpression or replication thereof. Selection and use of such vehiclesare well within the skill of the artisan. An expression vector includesvectors capable of expressing DNA operatively linked with regulatorysequences, such as promoter regions, that are capable of effectingexpression of such DNA molecules. Thus, an expression vector refers to arecombinant DNA or RNA construct, such as a plasmid, a phage,recombinant virus or other vector that, upon introduction into anappropriate host cell, results in expression of the cloned DNA.Appropriate expression vectors are well known to those of skill in theart and include those that are replicable in eukaryotic cells and/orprokaryotic cells and those that remain episomal or those whichintegrate into the host cell genome. Presently preferred plasmids forexpression of Gaussia luciferase, Renilla GFP and luciferase are thosethat are expressed in bacteria and yeast, such as those describedherein.

[0188] As used herein, a promoter region or promoter element refers to asegment of DNA or RNA that controls transcription of the DNA or RNA towhich it is operatively linked. The promoter region includes specificsequences that are sufficient for RNA polymerase recognition, bindingand transcription initiation. This portion of the promoter region isreferred to as the promoter. In addition, the promoter region includessequences that modulate this recognition, binding and transcriptioninitiation activity of RNA polymerase. These sequences may be cis actingor may be responsive to trans acting factors. Promoters, depending uponthe nature of the regulation, may be constitutive or regulated.Exemplary promoters contemplated for use in prokaryotes include thebacteriophage T7 and T3 promoters, and the like.

[0189] As used herein, operatively linked or operationally associatedrefers to the functional relationship of DNA with regulatory andeffector sequences of nucleotides, such as promoters, enhancers,transcriptional and translational stop sites, and other signalsequences. For example, operative linkage of DNA to a promoter refers tothe physical and functional relationship between the DNA and thepromoter such that the transcription of such DNA is initiated from thepromoter by an RNA polymerase that specifically recognizes, binds to andtranscribes the DNA. In order to optimize expression and/or in vitrotranscription, it may be necessary to remove, add or alter 5′untranslated portions of the clones to eliminate extra, potentialinappropriate alternative translation initiation (i.e., start) codons orother sequences that may interfere with or reduce expression, either atthe level of transcription or translation. Alternatively, consensusribosome binding sites (see, e.g., Kozak (1991) J. Biol. Chem.266:19867-19870) can be inserted immediately 5′ of the start codon andmay enhance expression. The desirability of (or need for) suchmodification may be empirically determined.

[0190] As used herein, to target a targeted agent, such as a luciferase,means to direct it to a cell that expresses a selected receptor or othercell surface protein by linking the agent to a such agent. Upon bindingto or interaction with the receptor or cell surface protein the targetedagent, can be reacted with an appropriate substrate and activatingagents, whereby bioluminescent light is produced and the tumorous tissueor cells distinguished from non-tumorous tissue.

[0191] As used herein, an effective amount of a compound for treating aparticular disease is an amount that is sufficient to ameliorate, or insome manner reduce the symptoms associated with the disease. Such amountmay be administered as a single dosage or may be administered accordingto a regimen, whereby it is effective. The amount may cure the diseasebut, typically, is administered in order to ameliorate the symptoms ofthe disease. Repeated administration may be required to achieve thedesired amelioration of symptoms.

[0192] As used herein, an effective amount of a conjugate for diagnosinga disease is an amount that will result in a detectable tissue. Thetissues are detected by visualization either without aid from a detectormore sensitive than the human eye, or with the use of a light source toexcite any fluorescent products.

[0193] As used herein, visualizable means detectable by eye,particularly during surgery under normal surgical conditions, or, ifnecessary, slightly dimmed light.

[0194] As used herein, pharmaceutically acceptable salts, esters orother derivatives of the conjugates include any salts, esters orderivatives that may be readily prepared by those of skill in this artusing known methods for such derivatization and that produce compoundsthat may be administered to animals or humans without substantial toxiceffects and that either are pharmaceutically active or are prodrugs.

[0195] As used herein, treatment means any manner in which the symptomsof a conditions, disorder or disease are ameliorated or otherwisebeneficially altered. Treatment also encompasses any pharmaceutical useof the compositions herein.

[0196] As used herein, amelioration of the symptoms of a particulardisorder by administration of a particular pharmaceutical compositionrefers to any lessening, whether permanent or temporary, lasting ortransient that can be attributed to or associated with administration ofthe composition.

[0197] As used herein, substantially pure means sufficiently homogeneousto appear free of readily detectable impurities as determined bystandard methods of analysis, such as thin layer chromatography (TLC),gel electrophoresis and high performance liquid chromatography (HPLC),used by those of skill in the art to assess such purity, or sufficientlypure such that further purification would not detectably alter thephysical and chemical properties, such as enzymatic and biologicalactivities, of the substance. Methods for purification of the compoundsto produce substantially chemically pure compounds are known to those ofskill in the art. A substantially chemically pure compound may, however,be a mixture of stereoisomers or isomers. In such instances, furtherpurification might increase the specific activity of the compound.

[0198] As used herein, a prodrug is a compound that, upon in vivoadministration, is metabolized or otherwise converted to thebiologically, pharmaceutically or therapeutically active form of thecompound. To produce a prodrug, the pharmaceutically active compound ismodified such that the active compound will be regenerated by metabolicprocesses. The prodrug may be designed to alter the metabolic stabilityor the transport characteristics of a drug, to mask side effects ortoxicity, to improve the flavor of a drug or to alter othercharacteristics or properties of a drug. By virtue of knowledge ofpharmacodynamic processes and drug metabolism in vivo, those of skill inthis art, once a pharmaceutically active compound is known, can designprodrugs of the compound (see, e.g., Nogrady (1985) Medicinal ChemistryA Biochemical Approach, Oxford University Press, New York, pages388-392).

[0199] As used herein, biological activity refers to the in vivoactivities of a compound or physiological responses that result upon invivo administration of a compound, composition or other mixture.Biological activity, thus, encompasses therapeutic effects andpharmaceutical activity of such compounds, compositions and mixtures.Biological activities may be observed in in vitro systems designed totest or use such activities. Thus, for purposes herein the biologicalactivity of a luciferase is its oxygenase activity whereby, uponoxidation of a substrate, light is produced.

[0200] As used herein, targeting agent refers to an agent thatspecifically or preferentially targets a linked targeted agent, aluciferin or luciferase, to a neoplastic cell or tissue.

[0201] As used herein, tumor antigen refers to a cell surface proteinexpressed or located on the surface of tumor cells.

[0202] As used herein, neoplastic cells include any type of transformedor altered cell that exhibits characteristics typical of transformedcells, such as a lack of contact inhibition and the acquisition oftumor-specific antigens. Such cells include, but are not limited toleukemic cells and cells derived from a tumor.

[0203] As used herein, neoplastic disease is any disease in whichneoplastic cells are present in the individual afflicted with thedisease. Such diseases include, any disease characterized as cancer.

[0204] As used herein, metastatic tumors refers to tumors that are notlocalized in one site.

[0205] As used herein, specialty tissue refers to non-tumorous tissuefor which information regarding location is desired. Such tissuesinclude, for example, endometriotic tissue, ectopic pregnancies, tissuesassociated with certain disorders and myopathies or pathologies.

[0206] As used herein, a receptor refers to a molecule that has anaffinity for a given ligand. Receptors may be naturally-occurring orsynthetic molecules. Receptors may also be referred to in the art asanti-ligands. As used herein, the receptor and anti-ligand areinterchangeable. Receptors can be used in their unaltered state or asaggregates with other species. Receptors may be attached, covalently ornoncovalently, or in physical contact with, to a binding member, eitherdirectly or indirectly via a specific binding substance or linker.Examples of receptors, include, but are not limited to: antibodies, cellmembrane receptors surface receptors and internalizing receptors,monoclonal antibodies and antisera reactive with specific antigenicdeterminants (such as on viruses, cells, or other materials), drugs,polynucleotides, nucleic acids, peptides, cofactors, lectins, sugars,polysaccharides, cells, cellular membranes, and organelles.

[0207] Examples of receptors and applications using such receptors,include but are not restricted to:

[0208] a) enzymes: specific transport proteins or enzymes essential tosurvival of microorganisms, which could serve as targets for antibiotic(ligand) selection;

[0209] b) antibodies: identification of a ligand-binding site on theantibody molecule that combines with the epitope of an antigen ofinterest may be investigated; determination of a sequence that mimics anantigenic epitope may lead to the development of vaccines of which theimmunogen is based on one or more of such sequences or lead to thedevelopment of related diagnostic agents or compounds useful intherapeutic treatments such as for auto-immune diseases

[0210] c) nucleic acids: identification of ligand, such as protein orRNA, binding sites;

[0211] d) catalytic polypeptides: polymers, preferably polypeptides,that are capable of promoting a chemical reaction involving theconversion of one or more reactants to one or more products; suchpolypeptides generally include a binding site specific for at least onereactant or reaction intermediate and an active functionality proximateto the binding site, in which the functionality is capable of chemicallymodifying the bound reactant (see, e.g., U.S. Pat. No. 5,215,899);

[0212] e) hormone receptors: determination of the ligands that bind withhigh affinity to a receptor is useful in the development of hormonereplacement therapies; for example, identification of ligands that bindto such receptors may lead to the development of drugs to control bloodpressure; and

[0213] f) opiate receptors: determination of ligands that bind to theopiate receptors in the brain is useful in the development ofless-addictive replacements for morphine and related drugs.

[0214] As used herein, antibody includes antibody fragments, such as Fabfragments, which are composed of a light chain and the variable regionof a heavy chain.

[0215] As used herein, an antibody conjugate refers to a conjugate inwhich the targeting agent is an antibody.

[0216] As used herein, antibody activation refers to the process wherebyactivated antibodies are produced. Antibodies are activated uponreaction with a linker, such as heterobifunctional reagent.

[0217] As used herein, a surgical viewing refers to any procedure inwhich an opening is made in the body of an animal. Such proceduresinclude traditional surgeries and diagnostic procedures, such aslaparoscopies and arthroscopic procedures.

[0218] As used herein, humanized antibodies refer to antibodies that aremodified to include “human” sequences of amino acids so thatadministration to a human will not provoke an immune response. Methodsfor preparation of such antibodies are known. For example, the hybridomathat expresses the monoclonal antibody is altered by recombinant DNAtechniques to express an antibody in which the amino acid composition ofthe non-variable regions is based on human antibodies. Computer programshave been designed to identify such regions.

[0219] As used herein, ATP, AMP, NAD+ and NADH refer to adenosinetriphosphate, adenosine monophosphate, nicotinamide adenine dinucleotide(oxidized form) and nicotinamide adenine dinucleotide (reduced form),respectively.

[0220] As used herein, production by recombinant means by usingrecombinant DNA methods means the use of the well known methods ofmolecular biology for expressing proteins encoded by cloned DNA.

[0221] As used herein, substantially identical to a product meanssufficiently similar so that the property of interest is sufficientlyunchanged so that the substantially identical product can be used inplace of the product.

[0222] As used herein equivalent, when referring to two sequences ofnucleic acids means that the two sequences in question encode the samesequence of amino acids or equivalent proteins. When “equivalent” isused in referring to two proteins or peptides, it means that the twoproteins or peptides have substantially the same amino acid sequencewith only conservative amino acid substitutions (see, e.g., Table 1,above) that do not substantially alter the activity or function of theprotein or peptide. When “equivalent” refers to a property, the propertydoes not need to be present to the same extent (e.g., two peptides canexhibit different rates of the same type of enzymatic activity), but theactivities are preferably substantially the same. “Complementary,” whenreferring to two nucleotide sequences, means that the two sequences ofnucleotides are capable of hybridizing, preferably with less than 25%,more preferably with less than 15%, even more preferably with less than5%, most preferably with no mismatches between opposed nucleotides.Preferably the two molecules will hybridize under conditions of highstringency.

[0223] As used herein: stringency of hybridization in determiningpercentage mismatch is as follows:

[0224] 1) high stringency: 0.1×SSPE, 0.1% SDS, 65° C.

[0225] 2) medium stringency: 0.2×SSPE, 0.1% SDS, 50° C.

[0226] 3) low stringency: 1.0×SSPE, 0.1% SDS, 50° C.

[0227] It is understood that equivalent stringencies may be achievedusing alternative buffers, salts and temperatures.

[0228] The term “substantially” identical or homologous or similarvaries with the context as understood by those skilled in the relevantart and generally means at least 70%, preferably means at least 80%,more preferably at least 90%, and most preferably at least 95% identity.The terms “homology” and “identity” are often used interchangeably. Ingeneral, sequences are aligned so that the highest order match isobtained (see, e.g.: Computational Molecular Biology, Lesk, A. M., ed.,Oxford University Press, New York, 1988; Biocomputing: Informatics andGenome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991; Carillo et al. (1988) SIAM J Applied Math 48:1073).

[0229] By sequence identity, the number of conserved amino acids aredetermined by standard alignment algorithms programs, and are used withdefault gap penalties established by each supplier. Substantiallyhomologous nucleic acid molecules would hybridize typically at moderatestringency or at high stringency all along the length of the nucleicacid of interest. Also contemplated are nucleic acid molecules thatcontain degenerate codons in place of codons in the hybridizing nucleicacid molecule.

[0230] Whether any two nucleic acid molecules have nucleotide sequencesthat are at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% “identical”can be determined using known computer algorithms such as the “FAST A”program, using for example, the default parameters as in Pearson et al.(1988) Proc. Natl. Acad. Sci. USA 85:2444 (other programs include theGCG program package (Devereux, J., et al., Nucleic Acids Research12(I):387 (1984)), BLASTP, BLASTN, FASTA (Atschul, S. F., et al., JMolec Biol 215:403 (1990); Guide to Huge Computers, Martin J. Bishop,ed., Academic Press, San Diego, 1994, and Carillo et al. (1988) SIAM JApplied Math 48:1073). For example, the BLAST function of the NationalCenter for Biotechnology Information database may be used to determineidentity. Other commercially or publicly available programs include,DNAStar “MegAlign” program (Madison, Wis.) and the University ofWisconsin Genetics Computer Group (UWG) “Gap” program (Madison Wis.)).Percent homology or identity of proteins and/or nucleic acid moleuclesmay be determined, for example, by comparing sequence information usinga GAP computer program (e.g., Needleman et al. (1970) J. Mol. Biol.48:443, as revised by Smith and Waterman ((1981) Adv. Appl. Math.2:482). Briefly, the GAP program defines similarity as the number ofaligned symbols (i.e., nucleotides or amino acids) which are similar,divided by the total number of symbols in the shorter of the twosequences. Default parameters for the GAP program may include: (1) aunary comparison matrix (containing a value of 1 for identities and 0for non-identities) and the weighted comparison matrix of Gribskov et al(1986) Nucl. Acids Res. 14:6745, as described by Schwartz and Dayhoff,eds., ATLAS OF PROTEIN SEQUENCE AND STRUCTURE, National BiomedicalResearch Foundation, pp. 353-358 (1979); (2) a penalty of 3.0 for eachgap and an additional 0.10 penalty for each symbol in each gap; and (3)no penalty for end gaps.

[0231] Therefore, as used herein, the term “identity” represents acomparison between a test and a reference polypeptide or polynucleotide.For example, a test polypeptide may be defined as any polypeptide thatis 90% or more identical to a reference polypeptide. As used herein, theterm at least “90% identical to” refers to percent identities from 90 to99.99 relative to the reference polypeptides. Identity at a level of 90%or more is indicative of the fact that, assuming for exemplificationpurposes a test and reference polynucleotide length of 100 amino acidsare compared. No more than 10% (i.e., 10 out of 100) amino acids in thetest polypeptide differs from that of the reference polypeptides.Similar comparisons may be made between a test and referencepolynucleotides. Such differences may be represented as point mutationsrandomly distributed over the entire length of an amino acid sequence orthey may be clustered in one or more locations of varying length up tothe maximum allowable, e.g. {fraction (10/100)} amino acid difference(approximately 90% identity). Differences are defined as nucleic acid oramino acid substitutions, or deletions. At level of homologies oridentities above about 85-90%, the result should be independent of theprogram and gap parameters set; such high levels of identity readily canbe assess, often without relying on software.

[0232] As used herein, primer refers to an oligonucleotide containingtwo or more deoxyribonucleotides or ribonucleotides, preferably morethan three, from which synthesis of a primer extension product can beinitiated. Experimental conditions conducive to synthesis include thepresence of nucleoside triphosphates and an agent for polymerization andextension, such as DNA polymerase, and a suitable buffer, temperatureand pH.

[0233] As used herein, a composition refers to any mixture. It may be asolution, a suspension, liquid, powder, a paste, aqueous, non-aqueous orany combination thereof.

[0234] As used herein, a combination refers to any association betweentwo or among more items.

[0235] As used herein, fluid refers to any composition that can flow.Fluids thus encompass compositions that are in the form of semi-solids,pastes, solutions, aqueous mixtures, gels, lotions, creams and othersuch compositions.

[0236] Examples of receptors and applications using such receptors,include but are not restricted to:

[0237] a) enzymes: specific transport proteins or enzymes essential tosurvival of microorganisms, which could serve as targets for antibiotic(ligand) selection;

[0238] b) antibodies: identification of a ligand-binding site on theantibody molecule that combines with the epitope of an antigen ofinterest may be investigated; determination of a sequence that mimics anantigenic epitope may lead to the development of vaccines of which theimmunogen is based on one or more of such sequences or lead to thedevelopment of related diagnostic agents or compounds useful intherapeutic treatments such as for auto-immune diseases

[0239] c) nucleic acids: identification of ligand, such as protein orRNA, binding sites;

[0240] d) catalytic polypeptides: polymers, preferably polypeptides,that are capable of promoting a chemical reaction involving theconversion of one or more reactants to one or more products; suchpolypeptides generally include a binding site specific for at least onereactant or reaction intermediate and an active functionality proximateto the binding site, in which the functionality is capable of chemicallymodifying the bound reactant (see, e.g., U.S. Pat. No. 5,215,899);

[0241] e) hormone receptors: determination of the ligands that bind withhigh affinity to a receptor is useful in the development of hormonereplacement therapies; for example, identification of ligands that bindto such receptors may lead to the development of drugs to control bloodpressure; and

[0242] f) opiate receptors: determination of ligands that bind to theopiate receptors in the brain is useful in the development ofless-addictive replacements for morphine and related drugs.

[0243] As used herein, complementary refers to the topologicalcompatibility or matching together of interacting surfaces of a ligandmolecule and its receptor. Thus, the receptor and its ligand can bedescribed as complementary, and furthermore, the contact surfacecharacteristics are complementary to each other.

[0244] As used herein, a ligand-receptor pair or complex formed when twomacromolecules have combined through molecular recognition to form acomplex.

[0245] As used herein, a substrate refers to any matrix that is usedeither directly or following suitable derivatization, as a solid supportfor chemical synthesis, assays and other such processes. Preferredsubstrates herein, are silicon substrates or siliconized substrates thatare derivitized on the surface intended for linkage of anti-ligands andligands and other macromolecules, including the fluorescent proteins,phycobiliproteins and other emission shifters.

[0246] As used herein, a matrix refers to any solid or semisolid orinsoluble support on which the molecule of interest, typically abiological molecule, macromolecule, organic molecule or biospecificligand is linked or contacted. Typically a matrix is a substratematerial having a rigid or semi-rigid surface. In many embodiments, atleast one surface of the substrate will be substantially flat, althoughin some embodiments it may be desirable to physically separate synthesisregions for different polymers with, for example, wells, raised regions,etched trenches, or other such topology. Matrix materials include anymaterials that are used as affinity matrices or supports for chemicaland biological molecule syntheses and analyses, such as, but are notlimited to: polystyrene, polycarbonate, polypropylene, nylon, glass,dextran, chitin, sand, pumice, polytetrafluoroethylene, agarose,polysaccharides, dendrimers, buckyballs, polyacrylamide,Kieselguhr-polyacrylamide non-covalent composite,polystyrene-polyacrylamide covalent composite, polystyrene-PEG(polyethyleneglycol) composite, silicon, rubber, and other materialsused as supports for solid phase syntheses, affinity separations andpurifications, hybridization reactions, immunoassays and other suchapplications.

[0247] As used herein, the attachment layer refers the surface of thechip device to which molecules are linked. Typically, the chip is asemiconductor device, which is coated on a least a portion of thesurface to render it suitable for linking molecules and inert to anyreactions to which the device is exposed. Molecules are linked eitherdirectly or indirectly to the surface, linkage may be effected byabsorption or adsorption, through covalent bonds, ionic interactions orany other interaction. Where necessary the attachment layer is adapted,such as by derivatization for linking the molecules.

[0248] B. Fluorescent Proteins

[0249] The GFP from Aequorea and that of the sea pansy Renillareniformis share the same chromophore, yet Aequorea GFP has twoabsorbance peaks at 395 and 475 nm, whereas Renilla GFP has only asingle absorbance peak at 498 nm, with about 5.5 fold greater monomerextinction coefficient the major 395 nm peak of the Aequorea protein(Ward, W. W. in Biohtminescence and Chemiluminescence (eds. DeLuca, M.A. & McElroy, W. D.) 235-242 (Academic Press, New York, 1981)). Thespectra of the isolated chromophore and denatured protein at neutral pHdo not match the spectra of either native protein (Cody, C. W. et al.(1993) Biochemistry 32:1212-1218).

[0250] 1. Green and Blue Fluorescent Proteins

[0251] As described herein, blue light is produced using the Renillaluciferase or the Aequorea photoprotein in the presence of Ca²⁺ and thecoelenterazine luciferin or analog thereof. This light can be convertedinto a green light if a green fluorescent protein (GFP) is added to thereaction. Green fluorescent proteins, which have been purified (see,e.g., Prasher et al. (1992) Gene 111:229-233) and also cloned (see,e.g., International PCT Application No. WO 95/07463, which is based onU.S. application Ser. No. 08/119,678 and U.S. application Ser. No.08/192,274, which are herein incorporated by reference), are used bycnidarians as energy-transfer acceptors. GFPs fluoresce in vivo uponreceiving energy from a luciferase-oxyluciferein excited-state complexor a Ca²⁺-activated photoprotein. The chromophore is modified amino acidresidues within the polypeptide. The best characterized GFPs are thoseof Aequorea and Renilla (see, e.g., Prasher et al. (1992) Gene111:229-233; Hart, et al. (1979) Biochemistry 18:2204-2210). Forexample, a green fluorescent protein (GFP) from Aequorea victoriacontains 238 amino acids, absorbs blue light and emits green light.Thus, inclusion of this protein in a composition containing the aequorinphotoprotein charged with coelenterazine and oxygen, can, in thepresence of calcium, result in the production of green light. Thus, itis contemplated that GFPs may be included in the bioluminescencegenerating reactions that employ the aequorin or Renilla luciferases orother suitable luciferase in order to enhance or alter color of theresulting bioluminescence.

[0252] 2. Renilla reniformis GFP

[0253] Purified Renilla reniformis GFP and muteins thereof are provided.Presently preferred Renilla GFP for use in the compositions herein isRenilla reniformis GFP having the sequence of amino acids set forth inSEQ ID No. 27. The Renilla GFP and GFP peptides can be isolated fromnatural sources or isolated from a prokaryotic or eukaryotic celltransfected with nucleic acid that encodes the Renilla GFP and/or GFPpeptides, such as those encoded by the sequences of nucleotides setforth in SEQ ID Nos. 23-25.

[0254] The encoding nucleic acid molecules are provided. Preferred arethose that encode the protein having the sequence of amino acids (SEQ IDNo. 27):

[0255] mdlaklglkevmptkinleglvgdhafsmegvgegnilegtqevkisvtkgapipfafdivsvafsygnraytgypeeisdyflqsfpegftyerniryqdggtaivksdisledgkfivnvdfkakdlrrmgpvmqqdivgmqpsyesmytnvtsvigeciiafklqtgkhftyhmrtvykskkpvetmplyhfiqhrlvktnvdtasgyvvqhetaiaahstikkiegsip,

[0256] and is preferably the sequence set forth in SEQ ID No. 26.

[0257] In particular, nucleic acid molecules encoding a Renillareniformis GFP having any of the following sequences are provided (seeSEQ ID Nos. 23-25): Renilla renformis GFP Clone-1GGCACGAGGGTTTCCTGACACAATAAAAACCTTTCAAATTGTTTCTCTGTAGCAGTAAGTATGGATCTCGCAAAACTTGGTTTGAAGGAAGTGATGCCTACTAAAATCAACTTAGAAGGACTGGTTGGCGACCACGCTTTCTCAATGGAAGGAGTTGGCGAAGGCAACATATTGGAAGGAACTCAAGAGGTGAAGATATCGGTAACAAAAGGCGCACCACTCCCATTCGCATTTGATATCGTATCTGTGGCTTTTTCATATGGGAACAGAGCTTATACCGGTTACCCAGAAGAAATTTCCGACTACTTCCTCCAGTCGTTTCCAGAAGGCTTTACTTACGAGAGAAACATTCGTTATCAAGATGGAGGAACTGCAATTGTTAAATCTGATATAAGCTTGGAAGATGGTAAATTCATAGTGAATGTAGACTTCAAAGCGAAGGATCTACGTCGCATGGGACCAGTCATGCAGCAAGACATCGTGGGTATGCAGCCATCGTATGAGTCAATGTACACCAATGTCACTTCAGTTATAGGGGAATGTATAATAGCATTCAAACTTCAAACTGGCAAGCATTTCACTTACCACATGAGGACAGTTTACAAATCAAAGAAGCCAGTGGAAACTATGCCATTGTATCATTTCATCCAGCATCGCCTCGTTAAGACCAATGTGGACACAGCCAGTGGTTACGTTGTGCAACACGAGACAGCAATTGCAGCGCATTCTACAATCAAAAAAATTGAAGGCTCTTTACCATAGATACCTGTACACAATTATTCTATGCACGTAGCATTTTTTTGGAAATATAAGTGGTATTGTTCAATAAAATATTAAATATAAAAAAAAAAAAAAAAAAAAAAAA; Renilla renformis GFP Clone-2GGCACGAGGCTGACACAATAAAAAACCTTTCAAATTGTTTCTCTGTAGCAGGAAGTATGGATCTCGCAAAACTTGGTTTGAAGGAAGTGATGCCTACTAAAATCAACTTAGAAGGACTGGTTGGCGACCACGCTTTCTCAATGGAAGGAGTTGGCGAAGGCAACATATTGGAAGGAACTCAAGAGGTGAAGATATCGGTAACAAAAGGCGCACCACTCCCATTCGCATTTGATATCGTATCTGTTGCTTTCTCATATGGGAACAGAGCTTATACTGGTTACCCAGAAGAAATTTCCGACTACTTCCTCCAGTCGTTTCCAGAAGGCTTTACTTACGAGAGAAACATTCGTTATCAAGATGGAGGAACTGCAATTGTTAAATCTGATATAAGCTTGGAAGATGGTAAATTCATAGTGAATGTAGACTTCAAAGCGAAGGATCTACGTCGCATGGGACCAGTCATGCAGCAAGACATCGTGGGTATGCAGCCATCGTATGAGTCAATGTACACCAATGTCACTTCAGTTATAGGGGAATGTATAATAGCATTCAAACTTCAAACTGGCAAACATTTCACTTACCACATGAGGACAGTTTACAAATCAAAGAAGCCAGTGGAAACTATGCCATTGTATCATTTCATCCAGCATCGCCTCGTTAAGACCAATGTGGACACAGCCAGTGGTTACGTTGTGCAACACGAGACAGCAATTGCAGCGCATTCTACAATCAAAAAAATTGAAGGCTCTTTACCATAGATATCTATACACAATTATTCTATGCACGTAGCATTTTTTTGGAAATATAAGTGGTATTGTTCAATAAAATATTAAATATAAAAAAAAAAAAAAAAAAAAAAA; and Renilla renformis GEP Clone-3GGCACGAGGGTTTCCTGACACAATAAAAACCTTTCAAATTGTTTCTCTGTAGCAGTAAGTATGGATCTCGCAAAACTTGGTTTGAAGGAAGTGATGCCTACTAAAATCAACTTAGAAGGACTGGTTGGCGACCACGCTTTCTCAATGGAAGGAGTTGGCGAAGGCAACATATTGGAAGGAACTCAAGAGGTGAAGATATCGGTAACAAAAGGCGCACCACTCCCATTCGCATTTGATATCGTATCTGTGGCTTTTTCATATGGGAACAGAGCTTATACCGGTTACCCAGAAGAAATTTCCGACTACTTCCTCCAGTCGTTTCCAGAAGGCTTTACTTACGAGAGAAACATTCGTTATCAAGATGGAGGAACTGCAATTGTTAAATCTGATATAAGCTTGGAAGATGGTAAATTCATAGTGAATGTAGACTTCAAAGCGAAGGATCTACGTCGCATGGGACCAGTGATGCAGCAAGACATCGTGGGTATGCAGCCATCGTATGAGTCAATGTACACCAATGTCACTTCAGTTATAGGGGAATGTATAATAGCATTCAAACTTCAAACTGGCAAGCATTTCACTTACCACATGAGGACAGTTTACAAATCAAAGAAGCCAGTGGAAACTATGCCATTGTATCATTTCATCCAGCATCGCCTCGTTAAGACCAATGTGGACACAGCCAGTGGTTACGTTGTGCAACACGAGACAGCAATTGCAGCGCATTCTACAATCAAAAAAATTGAAGGCTCTTTACCATAGATACCTGTACACAATTATTCTATGCACGTAGCATTTTTTTGGAAATATAAGTGGTATTGTTCAATAAAATATTAAATATATGCTTTTGCAAAAAAAAAAAAAAAAAAAAAA

[0258] are provided.

[0259] An exemplary mutein is set forth in SEQ ID No. 33, and humanizedcodon are set forth in SEQ ID No. 26.

[0260] Also contemplated are the coding portion of the sequence ofnucleotides that hybridize under moderate or high stringency to thesequence of nucleotides set forth above. particularly when using probesprovided herein, are provided. Probes derived from this nucleic acidthat can be used in methods provided herein to isolated GFPs from anyRenilla reniformis species. In an exemplary embodiment, nucleic acidencoding Renilla reniformis GFP is provided. This nucleic acid encodesthe sequence of amino acids set forth above.

[0261] GFPs, including the Renilla reniformis protein provided herein,are activated by blue light to emit green light and thus may be used inthe absence of luciferase and in conjunction with an external lightsource with novelty items (see U.S. Pat. Nos. 5,876,995, 6,152,358 and6,113,886) and in conjunction with bioluminescence generating system fornovelty items (see U.S. Pat. Nos. 5,876,995, 6,152,358 and 6,113,886),for tumor diagnosis (see, allowed co-pending U.S. application Ser. No.08/908,909) and in biochips (see, U.S. application Ser. No. 08/990,103,which is published as International PCT application No. WO 98/26277).

[0262]Renilla reniformis GFP is intended for use in any of the noveltyitems and combinations, such as the foods, including beverages, greetingcards, and toys, including bubble making toys, particularlybubble-making compositions or mixtures. Also of particular interest arethe use of these proteins in cosmetics, particularly face paints ormake-up, hair colorants or hair conditioners, mousses or other suchproducts and skin creams. Such systems are particularly of interestbecause no luciferase is needed to activate the photoprotein and becausethe proteins are non-toxic and safe to apply to the skin, hair, eyes andto ingest. These fluorescent proteins may also be used in addition tobioluminescence generating systems to enhance or create an array ofdifferent colors. Transgenic animals and plants that express the Renillareniformis GFP-encoding nucleic acid are also provided. Such animals andplants, include transgenic fish, transgenic worms for use, for example,as lures for fishing; transgenic animals, such as monkeys and rodentsfor research in which a marker gene is used, and transgenic animals asnovelty items and to produce glowing foods, such as ham, eggs, chicken,and other meats; transgenic plants in which the Renilla reniformis is amarker, and also transgenic plants that are novelty items, particuarlyornamental plants, such as glowing orchids, roses and other floweringplants.

[0263] The Renilla reniformis GFP may be used alone or in combinationwith bioluminescence generating systems to produce an array of colors.They may be used in combinations such that the color of, for example, abeverage changes over time, or includes layers of different colors. Thecloning and expression of Renilla reniformis GFP and uses thereof aredescribed below.

[0264] C. Bioluminescence Generating Systems and Components

[0265] The following is a description of bioluminescence generatingsystems and the components thereof. The Renilla reniformis GFP providedherein can be used alone for a variety of applications, and with anycompatible bioluminescence generating systems.

[0266] A bioluminescence-generating system refers to the components thatare necessary and sufficient to generate bioluminescence. These includea luciferase, luciferin and any necessary co-factors or conditions.Virtually any bioluminescent system known to those of skill in the artwill be amenable to use in the apparatus, systems, combinations andmethods provided herein. Factors for consideration in selecting abioluminescent-generating system, include, but are not limited to: thetargeting agent used in combination with the bioluminescence; the mediumin which the reaction is run; stability of the components, such astemperature or pH sensitivity; shelf life of the components;sustainability of the light emission, whether constant or intermittent;availability of components; desired light intensity; color of the light;and other such factors. Such bioluminescence generating systems areknown (see those described in U.S. Pat. Nos. 5,876,995, 6,152,358 and6,113,886).

[0267] 1. General Description

[0268] In general, bioluminescence refers to an energy-yielding chemicalreaction in which a specific chemical substrate, a luciferin, undergoesoxidation, catalyzed by an enzyme, a luciferase. Bioluminescentreactions are easily maintained, requiring only replenishment ofexhausted luciferin or other substrate or cofactor or other protein, inorder to continue or revive the reaction. Bioluminescence generatingreactions are well-known to those of skill in this art and any suchreaction may be adapted for use in combination with articles ofmanufacture as described herein.

[0269] There are numerous organisms and sources of bioluminescencegenerating systems, and some representative genera and species thatexhibit bioluminescence are set forth in the following table (reproducedin part from Hastings in (1995) Cell Physiology:Source Book, N.Sperelakis (ed.), Academic Press, pp 665-681): TABLE 2 Representativeluminous organism Type of Organism Representative genera BacteriaPhotobacterium Vibrio Xenorhabdus Mushrooms Panus, Armillaria PleurotusDinoflagellates Gonyaulax Pyrocystis Noctiluca Cnidaria (coelenterates)Jellyfish Aequorea Hydroid Obelia Sea Pansy Renilla CtenophoresMnemiopsis Beroe Annelids Earthworms Diplocardia Marine polychaetesChaetopterus, Phyxotrix Syllid fireworm Odontosyllis Molluscs LimpetLatia Clam Pholas Squid Heteroteuthis Heterocarpus Crustacea OstracodVargula (Cypridina) Shrimp (euphausids) Meganyctiphanes AcanthophyraOplophorus Gnathophausia Decapod Sergestes Copepods Insects Coleopterids(beetles) Firefly Photinus, Photuris Click beetles Pyrophorus Railroadworm Phengodes, Phrixothrix Diptera (flies) Arachnocampa EchinodermsBrittle stars Ophiopsila Sea cucumbers Laetmogone Anthozoans RenillaChordates Tunicates Pyrosoma Fish Cartilaginous Squalus Bony PonyfishLeiognathus Flashlight fish Photoblepharon Angler fish CryptopsarasMidshipman Porichthys Lantern fish Benia Shiny loosejaw AristostomiasHatchet fish Agyropelecus and other fish Pachystomias MalacosteusMidwater fish Cyclothone Neoscopelus Tarletonbeania

[0270] Other bioluminescent organisms contemplated for use herein areGonadostomias, Gaussia (copepods), Watensia, Halisturia, Vampire squid,Glyphus, Mycotophids (fish), Vinciguerria, Howella, Florenciella,Chaudiodus, Melanocostus and Sea Pens.

[0271] It is understood that a bioluminescence generating system may beisolated from natural sources, such as those in the above Table, or maybe produced synthetically. In addition, for uses herein, the componentsneed only be sufficiently pure so that mixture thereof, underappropriate reaction conditions, produces a glow so that cells andtissues can be visualized during a surgical procedure.

[0272] Thus, in some embodiments, a crude extract or merely grinding upthe organism may be adequate. Generally, however, substantially purecomponents are used. Also, components may be synthetic components thatare not isolated from natural sources. DNA encoding luciferases isavailable (see, e.g., SEQ ID Nos. 1-13) and has been modified (see,e.g., SEQ ID Nos. 3 and 10-13) and synthetic and alternative substrateshave been devised. The DNA listed herein is only representative of theDNA encoding luciferases that is available.

[0273] Any bioluminescence generating system, whether synthetic orisolated form natural sources, such as those set forth in Table 2,elsewhere herein or known to those of skill in the art, is intended foruse in the combinations, systems and methods provided herein.Chemiluminescence systems per se, which do not rely on oxygenases(luciferases) are not encompassed herein.

[0274] (a) Luciferases

[0275] Luciferases refer to any compound that, in the presence of anynecessary activators, catalyze the oxidation of a bioluminescencesubstrate (luciferin) in the presence of molecular oxygen, whether freeor bound, from a lower energy state to a higher energy state such thatthe substrate, upon return to the lower energy state, emits light. Forpurposes herein, luciferase is broadly used to encompass enzymes thatact catalytically to generate light by oxidation of a substrate and alsophotoproteins, such as aequorin, that act, though not strictlycatalytically (since such proteins are exhausted in the reaction), inconjunction with a substrate in the presence of oxygen to generatelight. These luciferases, including photoproteins, such as aequorin, areherein also included among the luciferases. These reagents include thenaturally-occurring luciferases (including photoproteins), proteinsproduced by recombinant DNA, and mutated or modified variants thereofthat retain the ability to generate light in the presence of anappropriate substrate, co-factors and activators or any other suchprotein that acts as a catalyst to oxidize a substrate, whereby light isproduced.

[0276] Generically, the protein that catalyzes or initiates thebioluminescent reaction is referred to as a luciferase, and theoxidizable substrate is referred to as a luciferin. The oxidizedreaction product is termed oxyluciferin, and certain luciferinprecursors are termed etioluciferin. Thus, for purposes hereinbioluminescence encompasses light produced by reactions that arecatalyzed by (in the case of luciferases that act enzymatically) orinitiated by (in the case of the photoproteins, such as aequorin, thatare not regenerated in the reaction) a biological protein or analog,derivative or mutant thereof.

[0277] For clarity herein, these catalytic proteins are referred to asluciferases and include enzymes such as the luciferases that catalyzethe oxidation of luciferin, emitting light and releasing oxyluciferin.Also included among luciferases are photoproteins, which catalyze theoxidation of luciferin to emit light but are changed in the reaction andmust be reconstituted to be used again. The luciferases may be naturallyoccurring or may be modified, such as by genetic engineering to improveor alter certain properties. As long as the resulting molecule retainsthe ability to catalyze the bioluminescent reaction, it is encompassedherein.

[0278] Any protein that has luciferase activity (a protein thatcatalyzes oxidation of a substrate in the presence of molecular oxygento produce light as defined herein) may be used herein. The preferredluciferases are those that are described herein or that have minorsequence variations. Such minor sequence variations include, but are notlimited to, minor allelic or species variations and insertions ordeletions of residues, particularly cysteine residues. Suitableconservative substitutions of amino acids are known to those of skill inthis art and may be made generally without altering the biologicalactivity of the resulting molecule. Such substitutions are preferablymade in accordance with those set forth in TABLE 1 as described above.

[0279] The luciferases may be obtained commercially, isolated fromnatural sources, expressed in host cells using DNA encoding theluciferase, or obtained in any manner known to those of skill in theart. For purposes herein, crude extracts obtained by grinding upselected source organisms may suffice. Since large quantities of theluciferase may be desired, isolation of the luciferase from host cellsis preferred. DNA for such purposes is widely available as are modifiedforms thereof.

[0280] Examples of luciferases include, but are not limited to, thoseisolated from the ctenophores Mnemiopsis (mnemiopsin) and Beroe ovata(berovin), those isolated from the coelenterates Aequorea (aequorin),Obelia (obelin), Pelagia, the Renilla luciferase, the luciferasesisolated from the mollusca Pholas (pholasin), the luciferases isolatedfrom fish, such as Aristostomias, Pachystomias and Poricthys and fromthe ostracods, such as Cypridina (also referred to as Vargula).Preferred luciferases for use herein are the Aequorin protein, Renillaluciferase and Cypridina (also called Vargula) luciferase (see, e.g.,SEQ ID Nos. 1, 2, and 4-13). Also, preferred are luciferases which reactto produce red and/or near infrared light. These include luciferasesfound in species of Aristostomias, such as A. scintillans, Pachystomias,Malacosteus, such as M. niger.

[0281] (b) Luciferins

[0282] The substrates for the reaction or for inclusion in theconjugates include any molecule(s) with which the luciferase reacts toproduce light. Such molecules include the naturally-occurringsubstrates, modified forms thereof, and synthetic substrates (see, e.g.,U.S. Pat. Nos. 5,374,534 and 5,098,828). Exemplary luciferins includethose described herein, as well as derivatives thereof, analogs thereof,synthetic substrates, such as dioxetanes (see, e.g., U.S. Pat. Nos.5,004,565 and 5,455,357), and other compounds that are oxidized by aluciferase in a light-producing reaction (see, e.g., U.S. Pat. Nos.5,374,534, 5,098,828 and 4,950,588). Such substrates also may beidentified empirically by selecting compounds that are oxidized inbioluminescent reactions.

[0283] (c) Activators

[0284] The bioluminescent generating systems also require additionalcomponents discussed herein and known to those of skill in the art. Allbioluminescent reactions require molecular oxygen in the form ofdissolved or bound oxygen. Thus, molecular oxygen, dissolved in water orin air or bound to a photoprotein, is the activator for bioluminescencereactions. Depending upon the form of the components, other activatorsinclude, but are not limited to, ATP (for firefly luciferase), flavinreductase (bacterial systems) for regenerating FMNH₂ from FMN, and Ca²⁺or other suitable metal ion (aequorin).

[0285] Most of the systems provided herein will generate light when theluciferase and luciferin are mixed and exposed to air or water. Thesystems that use photoproteins that have bound oxygen, such as aequorin,however, will require exposure to Ca²⁺ (or other suitable metal ion),which can be provided in the form of an aqueous composition of a calciumsalt. In these instances, addition of a Ca²⁺ (or other suitable metalion) to a mixture of luciferase (aequorin) and luciferin (such ascoelenterazine) will result in generation of light. The Renilla systemand other Anthozoa systems also require Ca²⁺ (or other suitable metalion).

[0286] If crude preparations are used, such as ground up Cypridina(shrimp) or ground fireflies, it may be necessary to add only water. Ininstances in which fireflies (or a firefly or beetle luciferase) areused the reaction may only require addition ATP. The precise componentswill be apparent, in light of the disclosure herein, to those of skillin this art or may be readily determined empirically.

[0287] It is also understood that these mixtures will also contain anyadditional salts or buffers or ions that are necessary for each reactionto proceed. Since these reactions are well-characterized, those of skillin the art will be able to determine precise proportions and requisitecomponents. Selection of components will depend upon the apparatus,article of manufacture and luciferase. Various embodiments are describedand exemplified herein; in view of such description, other embodimentswill be apparent.

[0288] (d) Reactions

[0289] In all embodiments, all but one component, either the luciferaseor luciferin, of a bioluminescence generating system will be mixed orpackaged with or otherwise combined. Since the result to be achieved isthe production of light visible to the naked eye for qualitative, notquantitative, diagnostic purposes, the precise proportions and amountsof components of the bioluminescence reaction need not be stringentlydetermined or met. They must be sufficient to produce light. Generally,an amount of luciferin and luciferase sufficient to generate a visibleglow is used; this amount can be readily determined empirically and isdependent upon the selected system and selected application. Wherequantitative measurements are required, more precision may be required.

[0290] For purposes herein, such amount is preferably at least theconcentrations and proportions used for analytical purposes by those ofskill in the such arts. Higher concentrations may be used if the glow isnot sufficiently bright. Alternatively, a microcarrier coupled to morethan one luciferase molecule linked to a targeting agent may be utilizedto increase signal output. Also because the conditions in which thereactions are used are not laboratory conditions and the components aresubject to storage, higher concentration may be used to overcome anyloss of activity. Typically, the amounts are 1 mg, preferably 10 mg andmore preferably 100 mg, of a luciferase per liter of reaction mixture or1 mg, preferably 10 mg, more preferably 100 mg. Compositions may containat least about 0.01 mg/l, and typically 0.1 mg/l, 1 mg/l, 10 mg/l ormore of each component on the item. The amount of luciferin is alsobetween about 0.01 and 100 mg/l, preferably between 0.1 and 10 mg/l,additional luciferin can be added to many of the reactions to continuethe reaction. In embodiments in which the luciferase acts catalyticallyand does not need to be regenerated, lower amounts of luciferase can beused. In those in which it is changed during the reaction, it also canbe replenished; typically higher concentrations will be selected. Rangesof concentration per liter (or the amount of coating on substrate theresults from contacting with such composition) of each component on theorder of 0.1 to 20 mg, preferably 0.1 to 10 mg, more preferably betweenabout 1 and 10 mg of each component will be sufficient. When preparingcoated substrates, as described herein, greater amounts of coatingcompositions containing higher concentrations of the luciferase orluciferin may be used.

[0291] Thus, for example, in presence of calcium, 5 mg of luciferin,such as coelenterazine, in one liter of water will glow brightly for atleast about 10 to 20 minutes, depending on the temperature of the water,when about 10 mgs of luciferase such as aequorin photoprotein luciferaseor luciferase from Renilla, is added thereto. Increasing theconcentration of luciferase, for example, to 100 mg/l, provides aparticularly brilliant display of light.

[0292] It is understood, that concentrations and amounts to be useddepend upon the selected bioluminescence generating system but these maybe readily determined empirically. Proportions, particularly those usedwhen commencing an empirical determination, are generally those used foranalytical purposes, and amounts or concentrations are at least thoseused for analytical purposes, but the amounts can be increased,particularly if a sustained and brighter glow is desired.

[0293] For purposes herein, Renilla reniformis GFP is added to thereaction in order to shift the spectrum of the generated light.

[0294] 2. The Renilla System

[0295] Renilla, also known as soft coral sea pansies, are members of theclass of coelenterates Anthozoa, which includes other bioluminescentgenera, such as Cavarnularia, Ptilosarcus, Stylatula, Acanthoptilum, andParazoanthus. Bioluminescent members of the Anthozoa genera containluciferases and luciferins that are similar in structure (see, e.g.,Cormier et al. (1973) J. Cell. Physiol. 81:291-298; see, also Ward etal. (1975) Proc. Natl. Acad. Sci. U.S.A. 72:2530-2534). The luciferasesand luciferins from each of these anthozoans crossreact with one anotherand produce a characteristic blue luminescence.

[0296] Renilla luciferase and the other coelenterate and ctenophoreluciferases, such as the aequorin photoprotein, use imidazopyrazinesubstrates, particularly the substrates generically calledcoelenterazine (see, formulae (I) and (II) of Section B.1.b, above).Other genera that have luciferases that use a coelenterazine include:squid, such as Chiroteuthis, Eucleoteuthis, Onychoteuthis, Watasenia,cuttlefish, Sepiolina; shrimp, such as Oplophorus, Acanthophyra,Sergestes, and Gnathophausia; deep-sea fish, such as Argyropelecus,Yarella, Diaphus, Gonadostomias and Neoscopelus.

[0297] Renilla luciferase does not, however, have bound oxygen, and thusrequires dissolved oxygen in order to produce light in the presence of asuitable luciferin substrate. Since Renilla luciferase acts as a trueenzyme (i.e., it does not have to be reconstituted for further use) theresulting luminescence can be long-lasting in the presence of saturatinglevels of luciferin. Also, Renilla luciferase is relatively stable toheat.

[0298] Renilla luciferases, DNA encoding Renilla reniformis luciferase,and use of the Renilla reniformis DNA to produce recombinant luciferase,as well as DNA encoding luciferase from other coelenterates, are wellknown and available (see, e.g., SEQ ID No. 1, U.S. Pat. Nos. 5,418,155and 5,292,658; see, also, Prasher et al. (1985) Biochem. Biophys. Res.Commun. 126:1259-1268; Cormier (1981) “Renilla and Aequoreabioluminescence” in Bioluminescence and Chemiluminescence, pp. 225-233;Charbonneau et al. (1979) J. Biol. Chem. 254:769-780; Ward et al. (1979)J. Biol. Chem. 254:781-788; Lorenz et al. (1981) Proc. Natl. Acad, Sci.U.S.A. 88: 4438-4442; Hori et al. (1977) Proc. Natl. Acad. Sci. U.S.A.74:4285-4287; Hori et al. (1975) Biochemistry 14:2371-2376; Hori et al.(1977) Proc. Natl. Acad. Sci. U.S.A. 74:4285-4287; Inouye et al. (1975)Jap. Soc. Chem. Lett. 141-144; and Matthews et al. (1979) Biochemistry16:85-91). The DNA encoding Renilla reniformis luciferase and host cellscontaining such DNA provide a convenient means for producing largequantities of Renilla reniformis enzyme, such as in those known to thoseof skill in the art (see, e.g., U.S. Pat. Nos. 5,418,155 and 5,292,658,which describe recombinant production of Renilla reniformis luciferase).

[0299] When used herein, the Renilla luciferase can be packaged inlyophilized form, encapsulated in a vehicle, either by itself or incombination with the luciferin substrate. Prior to use the mixture iscontacted with an aqueous composition, preferably a phosphate bufferedsaline pH 7-8; dissolved O₂ will activate the reaction. Finalconcentrations of luciferase in the glowing mixture will be on the orderof 0.01 to 1 mg/l or more. Concentrations of luciferin will be at leastabout 10⁻⁸ M, but 1 to 100 or more orders of magnitude higher to producea long lasting bioluminescence.

[0300] In certain embodiments herein, about 1 to 10 mg, or preferably2-5 mg, more preferably about 3 mg of coelenterazine will be used withabout 100 mg of Renilla luciferase. The precise amounts, of course canbe determined empirically, and, also will depend to some extent on theultimate concentration application. In particular, about addition ofabout 0.25 ml of a crude extract from the bacteria that express Renillato 100 ml of a suitable assay buffer and about 0.005 μg was sufficientto produce a visible and lasting glow (see, U.S. Pat. Nos. 5,418,155 and5,292,658, which describe recombinant production of Renilla reniformisluciferase).

[0301] Lyophilized mixtures, and compositions containing the Renillaluciferase are also provided. The luciferase or mixtures of theluciferase and luciferin may also be encapsulated into a suitabledelivery vehicle, such as a liposome, glass particle, capillary tube,drug delivery vehicle, gelatin, time release coating or other suchvehicle. The luciferase may also be linked to a substrate, such asbiocompatible materials.

[0302] 3. Ctenophore Systems

[0303] Ctenophores, such as Mnemiopsis (mnemiopsin) and Beroe ovata(berovin), and coelenterates, such as Aequorea (aequorin), Obelia(obelin) and Pelagia, produce bioluminescent light using similarchemistries (see, e.g., Stephenson et al. (1981) Biochimica etBiophysica Acta 678:65-75; Hart et al. (1979) Biochemistry 18:2204-2210;International PCT Application No. WO 94/18342, which is based on U.S.application Ser. No. 08/017,116, U.S. Pat. No. 5,486,455 and otherreferences and patents cited herein). The Aequorin and Renilla systemsare representative and are described in detail herein as exemplary andas among the presently preferred systems. The Aequorin and Renillasystems can use the same luciferin and produce light using the samechemistry, but each luciferase is different. The Aequorin luciferaseaequorin, as well as, for example, the luciferases mnemiopsin andberovin, is a photoprotein that includes bound oxygen and boundluciferin, requires Ca²⁺ (or other suitable metal ion) to trigger thereaction, and must be regenerated for repeated use; whereas, the Renillaluciferase acts as a true enzyme because it is unchanged during thereaction and it requires dissolved molecular oxygen.

[0304] 4. The Aequorin System

[0305] The aequorin system is well known (see, e.g., Tsuji et al. (1986)“Site-specific mutagenesis of the calcium-binding photoproteinaequorin,” Proc. Natl. Acad. Sci. USA 83:8107-8111; Prasher et al.(1985) “Cloning and Expression of the cDNA Coding for Aequorin, aBioluminescent Calcium-Binding Protein,” Biochemical and BiophysicalResearch Communications 126:1259-1268; Prasher et al. (1986) Methods inEnzymology 133:288-297; Prasher, et al. (1987) “Sequence Comparisons ofcDNAs Encoding for Aequorin Isotypes,” Biochemistry 26:1326-1332;Charbonneau et al. (1985) “Amino Acid Sequence of the Calcium-DependentPhotoprotein Aequorin,” Biochemistry 24:6762-6771; Shimomura et al.(1981) “Resistivity to denaturation of the apoprotein of aequorin andreconstitution of the luminescent photoprotein from the partiallydenatured apoprotein,” Biochem. J. 199:825-828; Inouye et al. (1989) J.Biochem. 105:473-477; Inouye et al. (1986) “Expression of ApoaequorinComplementary DNA in Escherichia coli,” Biochemistry 25:8425-8429;Inouye et al. (1985) “Cloning and sequence analysis of cDNA for theluminescent protein aequorin,” Proc. Natl. Acad. Sci. USA 82:3154-3158;Prendergast, et al. (1978) “Chemical and Physical Properties of Aequorinand the Green Fluorescent Protein Isolated from Aequorea forskalea” J.Am. Chem. Soc. 17:3448-3453; European Patent Application 0 540 064 A1;European Patent Application 0 226 979 A2, European Patent Application 0245 093 A1 and European Patent Application 0 245 093 B1; U.S. Pat. No.5,093,240; U.S. Pat. No. 5,360,728; U.S. Pat. No. 5,139,937; U.S. Pat.No. 5,422,266; U.S. Pat. No. 5,023,181; U.S. Pat. No. 5,162,227; and SEQID Nos. 5-13, which set forth DNA encoding the apoprotein; and a form,described in U.S. Pat. No. 5,162,227, European Patent Application 0 540064 A1 and Sealite Sciences Technical Report No. 3 (1994), iscommercially available from Sealite, Sciences, Bogart, Ga. asAQUALITE®).

[0306] This system is among the preferred systems for use herein. Aswill be evident, since the aequorin photoprotein includes noncovalentlybound luciferin and molecular oxygen, it is suitable for storage in thisform as a lyophilized powder or encapsulated into a selected deliveryvehicle. The system can be encapsulated into pellets, such as liposomesor other delivery vehicles. When used, the vehicles are contacted with acomposition, even tap water, that contains Ca²⁺ (or other suitable metalion), to produce a mixture that glows.

[0307] a. Aequorin and Related Photoproteins

[0308] The photoprotein, aequorin, isolated from the jellyfish,Aequorea, emits light upon the addition of Ca²⁺ (or other suitable metalion). The aequorin photoprotein, which includes bound luciferin andbound oxygen that is released by Ca²⁺, does not require dissolvedoxygen. Luminescence is triggered by calcium, which releases oxygen andthe luciferin substrate producing apoaqueorin.

[0309] The bioluminescence photoprotein aequorin is isolated from anumber of species of the jellyfish Aequorea. It is a 22 kilodalton (kD)molecular weight peptide complex (see, e.g., Shimomura et al. (1962) J.Cellular and Comp. Physiol. 59:233-238; Shimomura et al. (1969)Biochemistry 8:3991-3997; Kohama et al. (1971) Biochemistry10:4149-4152; and Shimomura et al. (1972) Biochemistry 11:1602-1608).The native protein contains oxygen and a heterocyclic compoundcoelenterazine, a luciferin, (see, below) noncovalently bound thereto.The protein contains three calcium binding sites. Upon addition of traceamounts Ca²⁺ (or other suitable metal ion, such as strontium) to thephotoprotein, it undergoes a conformational change that catalyzes theoxidation of the bound coelenterazine using the protein-bound oxygen.Energy from this oxidation is released as a flash of blue light,centered at 469 nm. Concentrations of calcium ions as low as 10⁻⁶ M aresufficient to trigger the oxidation reaction.

[0310] Naturally-occurring apoaequorin is not a single compound butrather is a mixture of microheterogeneous molecular species. Aequoriajellyfish extracts contain as many as twelve distinct variants of theprotein (see, e.g., Prasher et al. (187) Biochemistry 26:1326-1332;Blinks et al. (1975) Fed. Proc. 34:474). DNA encoding numerous forms hasbeen isolated (see, e.g., SEQ ID Nos. 5-9 and 13).

[0311] The photoprotein can be reconstituted (see, e.g., U.S. Pat. No.5,023,181) by combining the apoprotein, such as a protein recombinantlyproduced in E. coli, with a coelenterazine, such as a syntheticcoelenterazine, in the presence of oxygen and a reducing agent (see,e.g., Shimomura et al. (1975) Nature 256:236-238; Shimomura et al.(1981) Biochemistry J. 199:825-828), such as 2-mercaptoethanol, and alsoEDTA or EGTA (concentrations between about 5 to about 100 mM or higherfor applications herein) tie up any Ca²⁺ to prevent triggering theoxidation reaction until desired. DNA encoding a modified form of theapoprotein that does not require 2-mercaptoethanol for reconstitution isalso available (see, e.g., U.S. Patent No. U.S. Pat. No. 5,093,240). Thereconstituted photoprotein is also commercially available (sold, e.g.,under the trademark AQUALITE®, which is described in U.S. Pat. No.5,162,227).

[0312] The light reaction is triggered by adding Ca²⁺ at a concentrationsufficient to overcome the effects of the chelator and achieve the 10⁻⁶M concentration. Because such low concentrations of Ca²⁺ can trigger thereaction, for use in the methods herein, higher concentrations ofchelator may be included in the compositions of photoprotein.Accordingly, higher concentrations of added Ca²⁺ in the form of acalcium salt will be required. Precise amounts may be empiricallydetermined. For use herein, it may be sufficient to merely add water tothe photoprotein, which is provided in the form of a concentratedcomposition or in lyophilized or powdered form. Thus, for purposesherein, addition of small quantities of Ca²⁺, such as those present inphosphate buffered saline (PBS) or other suitable buffers or themoisture on the tissue to which the compositions are contacted, shouldtrigger the bioluminescence reaction.

[0313] Numerous isoforms of the aequorin apoprotein been identifiedisolated. DNA encoding these proteins has been cloned, and the proteinsand modified forms thereof have been produced using suitable host cells(see, e.g., U.S. Pat. Nos. 5,162,227, 5,360,728, 5,093,240; see, also,Prasher et al. (1985) Biophys. Biochem. Res. Commun. 126:1259-1268;Inouye et al. (1986) Biochemistry 25:8425-8429). U.S. Pat. No.5,093,240; U.S. Pat. No. 5,360,728; U.S. Pat. No. 5,139,937; U.S. Pat.No. 5,288,623; U.S. Pat. No. 5,422,266, U.S. Pat. No. 5,162,227 and SEQID Nos. 5-13, which set forth DNA encoding the apoprotein; and a form iscommercially available form Sealite, Sciences, Bogart, Ga. asAQUALITE®). DNA encoding apoaequorin or variants thereof is useful forrecombinant production of high quantities of the apoprotein. Thephotoprotein is reconstituted upon addition of the luciferin,coelenterazine, preferably a sulfated derivative thereof, or an analogthereof, and molecular oxygen (see, e.g., U.S. Pat. No. 5,023,181). Theapoprotein and other constituents of the photoprotein andbioluminescence generating reaction can be mixed under appropriateconditions to regenerate the photoprotein and concomitantly have thephotoprotein produce light. Reconstitution requires the presence of areducing agent, such as mercaptoethanol, except for modified forms,discussed below, that are designed so that a reducing agent is notrequired (see, e.g., U.S. Pat. No. 5,093,240).

[0314] For use herein, it is preferred aequorin is produced using DNA,such as that set forth in SEQ ID Nos. 5-13 and known to those of skillin the art or modified forms thereof. The DNA encoding aequorin isexpressed in a host cell, such as E. coli, isolated and reconstituted toproduce the photoprotein (see, e.g., U.S. Pat. Nos. 5,418,155,5,292,658, 5,360,728, 5,422,266, 5,162,227).

[0315] Of interest herein, are forms of the apoprotein that have beenmodified so that the bioluminescent activity is greater than unmodifiedapoaequorin (see, e.g., U.S. Pat. No. 5,360,728, SEQ ID Nos. 10-12).Modified forms that exhibit greater bioluminescent activity thanunmodified apoaequorin include proteins including sequences set forth inSEQ ID Nos. 10-12, in which aspartate 124 is changed to serine,glutamate 135 is changed to serine, and glycine 129 is changed toalanine, respectively. Other modified forms with increasedbioluminescence are also available.

[0316] For use in certain embodiments herein, the apoprotein and othercomponents of the aequorin bioluminescence generating system arepackaged or provided as a mixture, which, when desired is subjected toconditions under which the photoprotein reconstitutes from theapoprotein, luciferin and oxygen (see, e.g., U.S. Pat. No. 5,023,181;and U.S. Pat. No. 5,093,240). Particularly preferred are forms of theapoprotein that do not require a reducing agent, such as2-mercaptoethanol, for reconstitution. These forms, described, forexample in U.S. Pat. No. 5,093,240 (see, also Tsuji et al. (1986) Proc.Natl. Acad. Sci. U.S.A. 83:8107-8111), are modified by replacement ofone or more, preferably all three cysteine residues with, for exampleserine. Replacement may be effected by modification of the DNA encodingthe aequorin apoprotein, such as that set forth in SEQ ID No. 5, andreplacing the cysteine codons with serine.

[0317] The photoproteins and luciferases from related species, such asObelia are also contemplated for use herein. DNA encoding theCa²⁺-activated photoprotein obelin from the hydroid polyp Obelialongissima is known and available (see, e.g., Illarionov et al. (1995)Gene 153:273-274; and Bondar et al. (1995) Biochim. Biophys. Acta1231:29-32). This photoprotein can also be activated by Mn²⁺ (see, e.g.,Vysotski et al. (1995) Arch. Bioch. Biophys. 316:92-93, Vysotski et al.(1993) J. Biolumin. Chemilumin. 8:301-305).

[0318] In general for use herein, the components of the bioluminescenceare packaged or provided so that there is insufficient metal ions totrigger the reaction. When used, the trace amounts of triggering metalion, particularly Ca²⁺ is contacted with the other components. For amore sustained glow, aequorin can be continuously reconstituted or canbe added or can be provided in high excess.

[0319] b. Luciferin

[0320] The aequorin luciferin is coelenterazine and analogs therein,which include molecules including the structure (formula (I)):

[0321] in which R₁ is CH₂C₆H₅ or CH₃; R₂ is C₆H₅, and R₃ is p-C₆H₄OH orCH₃ or other such analogs that have activity. Preferred coelenterazinehas the structure in which R¹ is p-CH₂C₆H₄OH, R₂ is C₆H₅, and R₃ isp-C₆H₄OH, which can be prepared by known methods (see, e.g., Inouye etal. (1975) Jap. Chem. Soc., Chemistry Lttrs. pp 141-144; and Hart et al.(1979) Biochemistry 18:2204-2210). Among the preferred analogs, arethose that are modified, whereby the spectral frequency of the resultinglight is shifted to another frequency.

[0322] The preferred coelenterazine has the structure (formula (II)):

[0323] and sulfated derivatives thereof.

[0324] Another coelentratrazine has formula (V):

[0325] (see, Hart et al. (1979) Biochemistry 18:2204-2210). Using thisderivative in the presence of luciferase all of the light is in theultraviolet with a peak at 390 nm. Upon addition of GFP, all lightemitted is now in the visible range with a peak at 509 nm accompanied byan about 200-fold increase in the amount of light emitted. Viewed with acut-off filter of 470 nm, in the light yield in the absence of GFP wouldbe about zero, and would be detectable in the presence of GFP. Thisprovides the basis for an immunoassay described in the EXAMPLES.

[0326] The reaction of coelenterazine when bound to the aequorinphotoprotein with bound oxygen and in the presence of Ca²⁺ canrepresented as follows:

[0327] The photoprotein aequorin (which contains apoaequorin bound to acoelenterate luciferin molecule) and Renilla luciferase, discussedbelow, can use the same coelenterate luciferin. The aequorinphotoprotein catalyses the oxidation of coelenterate luciferin(coelenterazine) to oxyluciferin (coelenteramide) with the concomitantproduction of blue light (lambda_(max)=469 nm).

[0328] Importantly, the sulfate derivative of the coelenterate luciferin(lauryl-luciferin) is particularly stable in water, and thus may be usedin a coelenterate-like bioluminescent system. In this system, adenosinediphosphate (ADP) and a sulpha-kinase are used to convert thecoelenterazine to the sulphated form. Sulfatase is then used toreconvert the lauryl-luciferin to the native coelenterazine. Thus, themore stable lauryl-luciferin is used in the item to be illuminated andthe luciferase combined with the sulfatase are added to the luciferinmixture when illumination is desired.

[0329] Thus, the bioluminescent system of Aequorea is particularlysuitable for use in the methods herein. The particular amounts and themanner in which the components are provided depends upon the type ofneoplasia or specialty tissue to be visualized. This system can beprovided in lyophilized form, that will glow upon addition of Ca²⁺. Itcan be encapsulated, linked to microcarriers, such as microbeads, or inas a compositions, such as a solution or suspension, preferably in thepresence of sufficient chelating agent to prevent triggering thereaction. The concentration of the aequorin photoprotein will vary andcan be determined empirically. Typically concentrations of at least 0.1mg/l, more preferably at least 1 mg/l and higher, will be selected. Incertain embodiments, 1-10 mg luciferin/100 mg of luciferase will be usedin selected volumes and at the desired concentrations will be used.

[0330] 5. Crustacean, Particularly Cyrpidina Systems

[0331] The ostracods, such as Vargula serratta, hilgendorfii andnoctiluca are small marine crustaceans, sometimes called sea fireflies.These sea fireflies are found in the waters off the coast of Japan andemit light by squirting luciferin and luciferase into the water, wherethe reaction, which produces a bright blue luminous cloud, occurs. Thereaction involves only luciferin, luciferase and molecular oxygen, and,thus, is very suitable for application herein.

[0332] The systems, such as the Vargula bioluminescent systems, areparticularly preferred herein because the components are stable at roomtemperature if dried and powdered and will continue to react even ifcontaminated. Further, the bioluminescent reaction requires only theluciferin/luciferase components in concentrations as low as 1:40 partsper billion to 1:100 parts per billion, water and molecular oxygen toproceed. An exhausted system can renewed by addition of luciferin.

[0333] a. Vargula Luciferase

[0334] The Vargula luciferase is water soluble and is among thosepreferred for use in the methods herein. Vargula luciferase is a555-amino acid polypeptide that has been produced by isolation fromVargula and also using recombinant technology by expressing the DNA insuitable bacterial and mammalian host cells (see, e.g., Thompson et al.(1989) Proc. Natl. Acad. Sci. U.S.A. 86:6567-6571; Inouye et al. (1992)Proc. Natl. Acad. Sci. U.S.A. 89:9584-9587; Johnson et al. (1978)Methods in Enzymology LVII:331-349; Tsuji et al. (1978) Methods Enzymol.57:364-72; Tsuji (1974) Biochemistry 13:5204-5209; Japanese PatentApplication No. JP 3-30678 Osaka; and European Patent Application No. EP0 387 355 A1).

(1) Purification From Cypridina

[0335] Methods for purification of Vargula (Cypridina) luciferase arewell known. For example, crude extracts containing the active can bereadily prepared by grinding up or crushing the Vargula shrimp. In otherembodiments, a preparation of Cypridina hilgendorfi luciferase can beprepared by immersing stored frozen C. hilgendorfi in distilled watercontaining, 0.5-5.0 M salt, preferably 0.5-2.0 M sodium or potassiumchloride, ammonium sulfate, at 0-30° C., preferably 0-10° C., for 1-48hr, preferably 10-24 hr, for extraction followed by hydrophobicchromatography and then ion exchange or affinity chromatography (TORAYIND INC, Japanese patent application JP 4258288, published Sep. 14,1993; see, also, Tsuji et al. (1978) Methods Enzymol. 57:364-72 forother methods).

(2) Preparation by Recombinant Methods

[0336] The luciferase is preferably produced by expression of cloned DNAencoding the luciferase (European Patent Application No. 0 387 355 A1;International PCT Application No. WO 95/001542; see, also SEQ ID No. 5,which sets forth the sequence from Japanese Patent Application No. JP3-30678 and Thompson et al. (1989) Proc. Natl. Acad. Sci. U.S.A.86:6567-6571) DNA encoding the luciferase or variants thereof isintroduced into E. coli using appropriate vectors and isolated usingstandard methods.

[0337] b. Vargula Luciferin

[0338] The natural luciferin is a substituted imidazopyrazine nucleus,such a compound of formula (III):

[0339] The luciferin can be isolated from ground dried Vargula byheating the extract, which destroys the luciferase but leaves theluciferin intact (see, e.g., U.S. Pat. No. 4,853,327).

[0340] Analogs thereof and other compounds that react with theluciferase in a light producing reaction also may be used.

[0341] Other bioluminescent organisms that have luciferases that canreact with the Vargula luciferin include, the genera Apogon,Parapriacanthus and Porichthys.

[0342] c. Reaction

[0343] The luciferin upon reaction with oxygen forms a dioxetanoneintermediate (which includes a cyclic peroxide similar to the fireflycyclic peroxide molecule intermediate). In the final step of thebioluminescent reaction, the peroxide breaks down to form CO₂ and anexcited carbonyl. The excited molecule then emits a blue to blue-greenlight.

[0344] The optimum pH for the reaction is about 7. For purposes herein,any pH at which the reaction occurs may be used. The concentrations ofreagents are those normally used for analytical reactions or higher(see, e.g., Thompson et al. (1990) Gene 96:257-262). Typicallyconcentrations of the luciferase between 0.1 and 10 mg/l, preferably 0.5to 2.5 mg/l will be used. Similar concentrations or higherconcentrations of the luciferin may be used.

[0345] 6. Insect Bioluminescent Systems Including Fireflies, ClickBeetles, and Other Insect System

[0346] The biochemistry of firefly bioluminescence was the firstbioluminescent system to be characterized (see, e.g., Wienhausen et al.(1985) Photochemistry and Photobiology 42:609-611; McElroy et al. (1966)in Molecular Architecture in cell Physiology, Hayashi et al., eds.Prentice Hall, Inc., Englewood Cliffs, N.J., pp. 63-80) and it iscommercially available (e.g., from Promega Corporation, Madison, Wis.,see, e.g., Leach et al. (1986) Methods in Enzymology 133:51-70, esp.Table 1). Luciferases from different species of fireflies areantigenically similar. These species include members of the generaPhotinus, Photurins and Luciola. Further, the bioluminescent reactionproduces more light at 30° C. than at 20° C., the luciferase isstabilized by small quantities of bovine albumin serum, and the reactioncan be buffered by tricine.

[0347] a. Luciferase

[0348] DNA clones encoding luciferases from various insects and the useto produce the encoded luciferase is well known. For example, DNA clonesthat encode luciferase from Photinus pyralis, Luciola cruciata (see,e.g., de Wet et al. (1985) Proc. Natl. Acad. Sci. U.S.A. 82:7870-7873;de We et al. (1986) Methods in Enzymology 133:3; U.S. Pat. No.4,968,613, see, also SEQ ID No. 3) are available. The DNA has also beenexpressed in Saccharomyces (see, e.g., Japanese Application No. JP63317079, published Dec. 26, 1988, KIKKOMAN CORP) and in tobacco.

[0349] In addition to the wild-type luciferase modified insectluciferases have been prepared. For example, heat stable luciferasemutants, DNA-encoding the mutants, vectors and transformed cells forproducing the luciferases are available. A protein with 60% amino acidsequence homology with luciferases from Photinus pyralis, Luciolamingrelica, L. cruciata or L. lateralis and having luciferase activityis available (see, e.g., International PCT Application No. WO 95/25798).It is more stable above 30° C. than naturally-occurring insectluciferases and may also be produced at 37° C. or above, with higheryield.

[0350] Modified luciferases that generate light at different wavelengths(compared with native luciferase), and thus, may be selected for theircolor-producing characteristics. For example, synthetic mutant beetleluciferase(s) and DNA encoding such luciferases that producebioluminescence at a wavelength different from wild-type luciferase areknown (Promega Corp, International PCT Application No. WO 95/18853,which is based on U.S. application Ser. No. 08/177,081). The mutantbeetle luciferase has an amino acid sequence differing from that of thecorresponding wild-type Luciola cruciata (see, e.g., U.S. Pat. Nos.5,182,202, 5,219,737, 5,352,598, see, also SEQ ID No.3) by asubstitution(s) at one or two positions. The mutant luciferase producesa bioluminescence with a wavelength of peak intensity that differs by atleast 1 nm from that produced by wild-type luciferases.

[0351] Other mutant luciferases can be produced. Mutant luciferases withthe amino acid sequence of wild-type luciferase, but with at least onemutation in which valine is replaced by isoleucine at the amino acidnumber 233, valine by isoleucine at 239, serine by asparagine at 286,glycine by serine at 326, histidine by tyrosine at 433 or proline byserine at 452 are known (see, e.g., U.S. Pat. Nos. 5,219,737, and5,330,906). The luciferases are produced by expressing DNA-encoding eachmutant luciferase in E. coli and isolating the protein. Theseluciferases produce light with colors that differ from wild-type. Themutant luciferases catalyze luciferin to produce red (λ 609 nm and 612nm), orange (λ595 and 607 nm) or green (λ 558 nm) light. The otherphysical and chemical properties of mutant luciferase are substantiallyidentical to native wild type-luciferase. The mutant luciferase has theamino acid sequence of Luciola cruciata luciferase with an alterationselected from Ser 286 replaced by Asn, Gly 326 replaced by Ser, His 433replaced by Tyr or Pro 452 replaced by Ser. Thermostable luciferases arealso available (see, e.g., U.S. Pat. No. 5,229,285; see, alsoInternational PCT Application No. WO 95/25798, which provides Photinusluciferase in which the glutamate at position 354 is replaced lysine andLuciola luciferase in which the glutamate at 356 is replaced withlysine).

[0352] These mutant luciferases as well as the wild type luciferases canbe used in combination with the GFPs provided herein particularly ininstances when a variety of colors are desired or when stability athigher temperatures is desired.

[0353] b. Luciferin

[0354] The firefly luciferin is a benzothiazole:

[0355] Analogs of this luciferin and synthetic firefly luciferins arealso known to those of skill in art (see, e.g., U.S. Pat. No. 5,374,534and 5,098,828). These include compounds of formula (IV) (see, U.S. Pat.No. 5,098,828):

[0356] in which:

[0357] R¹ is hydroxy, amino, linear or branched C₁-C₂₀ alkoxy, C₂-C₂₀alkyenyloxy, an L-amino acid radical bond via the α-amino group, anoligopeptide radical with up to ten L-amino acid units linked via theα-amino group of the terminal unit;

[0358] R² is hydrogen, H₂PO₃, HSO₃, unsubstituted or phenyl substitutedlinear or branched C₁-C₂₀ alkyl or C₂-C₂₀alkenyl, aryl containing 6 to18 carbon atoms, or R³—C(O)—; and

[0359] R³ is an unsubstituted or phenyl substituted linear or branchedC₁-C₂₀ alkyl or C₂-C₂₀alkenyl, aryl containing 6 to 18 carbon atoms, anucleotide radical with 1 to 3 phosphate groups, or a glycosidicallyattached mono- or disaccharide, except when formula (IV) is aD-luciferin or D-luciferin methyl ester.

[0360] Modified luciferins that have been modified to produce light ofshifted frequencies are known to those of skill in the art.

[0361] c. Reaction

[0362] The reaction catalyzed by firefly luciferases and related insectluciferases requires ATP, Mg²⁺ as well as molecular oxygen. Luciferinmust be added exogenously. Firefly luciferase catalyzes the fireflyluciferin activation and the subsequent steps leading to the excitedproduct. The luciferin reacts with ATP to form a luciferyl adenylateintermediate. This intermediate then reacts with oxygen to form a cyclicluciferyl peroxy species, similar to that of the coelenterateintermediate cyclic peroxide, which breaks down to yield CO₂ and anexcited state of the carbonyl product. The excited molecule then emits ayellow light; the color, however, is a function of pH. As the pH islowered the color of the bioluminescence changes from yellow-green tored.

[0363] Different species of fireflies emit different colors ofbioluminescence so that the color of the reaction will be dependent uponthe species from which the luciferase is obtained. Additionally, thereaction is optimized at pH 7.8.

[0364] Addition of ATP and luciferin to a reaction that is exhaustedproduces additional light emission. Thus, the system, once established,is relatively easily maintained. Therefore, it is highly suitable foruse herein in embodiments in which a sustained glow is desired.

[0365] 7. Other Systems

[0366] Numerous other systems are known and have been described indetail for example in U.S. Pat. Nos. 5,876,995, 6,152,358 and6,113,886).

[0367] a. Bacterial Systems

[0368] Luminous bacteria typically emit a continuous light, usuallyblue-green. When strongly expressed, a single bacterium may emit 10⁴ to10⁵ photons per second. Bacterial bioluminescence systems include, amongothers, those systems found in the bioluminescent species of the generaPhotobacterium, Vibrio and Xenorhabdus. These systems are well known andwell characterized (see, e.g., Baldwin et al. (1984) Biochemistry23:3663-3667; Nicoli et al. (1974) J. Biol. Chem. 249:2393-2396; Welcheset al. (1981) Biochemistry 20:512-517; Engebrecht et al. (1986) Methodsin Enzymology 133:83-99; Frackman et al. (1990) J. of Bacteriology172:5767-5773; Miyamoto et al. (1986) Methods in Enzymology 133:70; U.S.Pat. No. 4,581,335).

[0369] (1) Luciferases

[0370] Bacterial luciferase, as exemplified by luciferase derived fromVibrio harveyi (EC 1.14.14.3, alkanol reduced-FMN-oxygen oxidoreductase1-hydroxylating, luminescing), is a mixed function oxidase, formed bythe association of two different protein subunits α and β. The α-subunithas an apparent molecular weight of approximately 42,000 kD and theβ-subunit has an apparent molecular weight of approximately 37,000 kD(see, e.g., Cohn et al. (1989) Proc. Natl. Acad. Sci. U.S.A.90:102-123). These subunits associate to form a 2-chain complexluciferase enzyme, which catalyzes the light emitting reaction ofbioluminescent bacteria, such as Vibrio harveyi (U.S. Pat. No.4,581,335; Belas et al. (1982) Science 218:791-793), Vibrio fischeri(Engebrecht et al. (1983) Cell 32:773-781; Engebrecht et al. (1984)Proc. Natl. Acad. Sci. U.S.A. 81:4154-4158) and other marine bacteria.

[0371] Bacterial luciferase genes have been cloned (see, e.g., U.S. Pat.No. 5,221,623; U.S. Pat. No. 4,581,335; European Patent Application No.EP 386 691 A). Plasmids for expression of bacterial luciferase, such asVibrio harveyi, include pFIT001 (NRRL B-18080), pPALE001 (NRRL B-18082)and pMR19 (NRRL B-18081)) are known. For example the sequence of theentire lux regulon from Vibiro fisheri has been determined (Baldwin etal. (1984), Biochemistry 23:3663-3667; Baldwin et al. (1981) Biochem.20:512-517; Baldwin et al. (1984) Biochem. 23:3663-3667; see, also,e.g., U.S. Pat. Nos. 5,196,318, 5,221,623, and 4,581,335). This regulonincludes luxI gene, which encodes a protein required for autoinducersynthesis (see, e.g., Engebrecht et al. (1984) Proc. Natl. Acad. Sci.U.S.A. 81:4154-4158), the luxC, luxD, and luxE genes, which encodeenzymes that provide the luciferase with an aldehyde substrate, and theluxA and luxB genes, which encode the alpha and beta subunits of theluciferase.

[0372] Lux genes from other bacteria have also been cloned and areavailable (see, e.g., Cohn et al. (1985) J. Biol. Chem. 260:6139-6146;U.S. Pat. No. 5,196,524, which provides a fusion of the luxA and luxBgenes from Vibrio harveyl). Thus, luciferase alpha and betasubunit-encoding DNA is provided and can be used to produce theluciferase. DNA encoding the α (1065 bp) and β (984 bp) subunits, DNAencoding a luciferase gene of 2124 bp, encoding the alpha and betasubunits, a recombinant vector containing DNA encoding both subunits anda transformed E. coli and other bacterial hosts for expression andproduction of the encoded luciferase are available. In addition,bacterial luciferases are commercially available.

[0373] (2) Luciferins

[0374] Bacterial luciferins include:

[0375] in which the tetradecanal with reduced flavin mononucleotide areconsidered luciferin since both are oxidized during the light emittingreaction.

[0376] (3) Reactions

[0377] The bacterial systems require, in addition to reduced flavin,five polypeptides to complete the bioluminescent reaction: two subunits,α and β, of bacterial luciferin and three units of a fatty acidreductase system complex, which supplies the tetradecanal aldehyde.Examples of bacterial bioluminescent systems useful in the apparatus andmethods provided herein include those derived from Vibrio fisheri andVibrio harveyi. One advantage to this system is its ability to operateat cold temperatures; certain surgical procedures are performed bycooling the body to lower temperatures.

[0378] Bacterial luciferase catalyzes the flavin-mediated hydroxylationof a long-chain aldehyde to yield carboxylic acid and an excited flavin;the flavin decays to ground state with the concomitant emission of bluegreen light (λ_(max)=490 nm; see, e.g., Legocki et al. (1986) Proc.Natl. Acad. Sci. USA 81:9080; see U.S. Pat. No. 5,196,524):

[0379] The reaction can be initiated by contacting reduced flavinmononucleotide (FMNH₂) with a mixture of the bacterial luciferase,oxygen, and a long-chain aldehyde, usually n-decyl aldehyde.

[0380] DNA encoding luciferase from the fluorescent bacteriumAlteromonas hanedai is known (CHISSO CORP; see, also, Japaneseapplication JP 7222590, published Aug. 22, 1995). The reduced flavinmononucleotide (FMNH₂; luciferin) reacts with oxygen in the presence ofbacterial luciferase to produce an intermediate peroxy flavin. Thisintermediate reacts with a long-chain aldehyde (tetradecanal) to formthe acid and the luciferase-bound hydroxy flavin in its excited state.The excited luciferase-bound hydroxy flavin then emits light anddissociates from the luciferase as the oxidized flavin mononucleotide(FMN) and water. In vivo FMN is reduced again and recycled, and thealdehyde is regenerated from the acid.

[0381] Flavin reductases have been cloned (see, e.g., U.S. Pat. No.5,484,723; see, SEQ ID No. 14 for a representative sequence from thispatent). These as well as NAD(P)H can be included in the reaction toregenerate FMNH₂ for reaction with the bacterial luciferase and longchain aldehyde. The flavin reductase catalyzes the reaction of FMN,which is the luciferase reaction, into FMNH₂; thus, if luciferase andthe reductase are included in the reaction system, it is possible tomaintain the bioluminescent reaction. Namely, since the bacterialluciferase turns over many times, bioluminescence continues as long as along chain aldehyde is present in the reaction system.

[0382] The color of light produced by bioluminescent bacteria alsoresults from the participation of a protein blue-florescent protein(BFP) in the bioluminescence reaction. This protein, which is well known(see, e.g., Lee et al. (1978) Methods in Enzymology LVII:226-234), mayalso be added to bacterial bioluminescence reactions in order to cause ashift in the color.

[0383] b. Dinoflagellate Bioluminescence Generating Systems

[0384] In dinoflagellates, bioluminescence occurs in organelles termedscintillons. These organelles are outpocketings of the cytoplasm intothe cell vacuole. The scintillons contain only dinoflagellate luciferaseand luciferin (with its binding protein), other cytoplasmic componentsbeing somehow excluded. The dinoflagellate luciferin is a tetrapyrrolerelated to chlorophyll:

[0385] or an analog thereof.

[0386] The luciferase is a 135 kD single chain protein that is active atpH 6.5, but inactive at pH 8 (see, e.g., Hastings (1981) Bioluminescenceand Chemiluminescence, DeLuca et al., eds. Academic Press, NY,pp.343-360). Luminescent activity can be obtained in extracts made at pH8 by simply shifting the pH from 8 to 6. This occurs in soluble andparticulate fractions. Within the intact scintillon, the luminescentflash occurs for ˜100 msec, which is the duration of the flash in vivo.In solution, the kinetics are dependent on dilution, as in any enzymaticreaction. At pH 8, the luciferin is bound to a protein (luciferinbinding protein) that prevents reaction of the luciferin with theluciferase. At pH 6, however, the luciferin is released and free toreact with the enzyme.

[0387] D. Isolation and Identification of Nucleic Acids EncodingLuciferases and GFPs

[0388] Nucleic acid encoding bioluminescent proteins are provided.Particularly, nucleic acid encoding Renilla reniformis GFP is provided.

[0389] 1. Isolation of Specimens of the Genus Renilla

[0390] Specimens of Renilla are readily available from the oceans of theworld, including the Gulf of Mexico, Pacific Ocean and Atlantic Ocean.Renilla typically live on the ocean bottom at about 30 to 100 feet deepand can be easily collected by dragging. For example, specimens of R.kollikeri can be obtained off the coast of California or Baja, Mexico.Alternatively, live specimens of Renilla may be purchased from acommercial supplier (e.g., Gulf Marine Incorporated, Panacea, Fla.).Upon capture or receipt, the specimens are washed thoroughly and mayalso be dissected to enrich for light-emitting tissues. The wholeorganisms or dissected tissues are then snap frozen and stored in liquidnitrogen.

[0391] As described in detail in the examples below, the frozen tissueswere used as a source to isolate nucleic acids encoding Renilla mulleriGFP and luciferase (e.g., see SEQ ID No. 15 and SEQ ID No. 17,respectively).

[0392] 2. Preparation of Renilla cDNA Expression Libraries

[0393] Renilla cDNA expression libraries may be prepared from intact RNAfollowing the methods described herein or by other methods known tothose of skill the art (e.g., see Sambrook et al. (1989) MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.; U.S. Pat. No. 5,292,658).

[0394] Typically, the preparation of cDNA libraries includes theisolation of polyadenylated RNA from the selected organism followed bysingle-strand DNA synthesis using reverse transcriptase, digestion ofthe RNA strand of the DNA/RNA hybrid and subsequent conversion of thesingle-stranded DNA to double stranded cDNA.

[0395] a. RNA Isolation and cDNA Synthesis

[0396] Whole Renilla or dissected Renilla tissues can be used a sourceof total cytoplasmic RNA for the preparation of Renilla cDNA. Totalintact RNA can be isolated from crushed Renilla tissue, for example, byusing a modification of methods generally known in the art (e.g., seeChirgwin et al. (1970) Biochemistry 18:5294-5299). After isolating totalcellular RNA, polyadenylated RNA species are then easily separated fromthe nonpolyadenylated species using affinity chromatography onoligodeoxythymidylate cellulose columns, (e.g., as described by Aviv etal., (1972) Proc. Natl. Acad. Sci. U.S.A. 69:1408).

[0397] The purified Renilla polyA-mRNA is then subjected to a cDNAsynthesis reaction to generate a cDNA library from total polyA-mRNA.Briefly, reverse transcriptase is used to extend an annealed polydTprimer to generate an RNA/DNA duplex. The RNA strand is then digestedusing an RNase, e.g., RNase H, and following second-strand synthesis,the cDNA molecules are blunted-ended with S1 nuclease or otherappropriate nuclease. The resulting double-stranded cDNA fragments canbe ligated directly into a suitable expression vector or, alternatively,oligonucleotide linkers encoding restriction endonuclease sites can beligated to the 5′-ends of the cDNA molecules to facilitate cloning ofthe cDNA fragments.

[0398] b. Construction of cDNA Expression Libraries

[0399] The best characterized vectors for the construction of cDNAexpression libraries are lambda vectors. Lambda-based vectors toleratecDNA inserts of about 12 kb and provide greater ease in libraryscreening, amplification and storage compared to standard plasmidvectors. Presently preferred vectors for the preparation of Renilla cDNAexpression libraries are the Lambda, Uni-Zap, Lambda-Zap II orLambda-ZAP Express/EcoRI/XhoI vectors, which are known to those of skillin the art (e.g., see U.S. Pat. No. 5,128,256), and are alsocommercially available (Stratagene, La Jolla, Calif.).

[0400] Generally, the Lambda-Zap vectors combine the high efficiency ofa bacteriophage lambda vector systems with the versatility of a plasmidsystem. Fragments cloned into these vectors can be automatically excisedusing a helper phage and recircularized to generate subclones in thepBK-derived phagemid. The pBK phagemid carries the neomycin-resistancegene for selection in bacteria and G418 selection in eukaryotic cells ormay contain the β-lactamase resistance gene. Expression of therecombinant polypeptide is under the control of the lacZ promoter inbacteria and the CMV promoter in eukaryotes.

[0401] More specifically, these lambda-based vectors are composed of aninitiator-terminator cassette containing the plasmid system, e.g., a pBKBluescript derivative (Stratagene, San Diego), bracketed by the rightand left arm of the bacteriophage lambda. The lambda arms allow forefficient packaging of replicated DNA whereas the excisableinitiator-terminator cassette allows for easy cloning of the cDNAfragments and the generation of a plasmid library without the need foradditional subcloning.

[0402] When used herein, cDNA fragments are inserted into the multiplecloning site contained within the initiator-terminator cassette of theLambda-Zap vector to create a set of cDNA expression vectors. The set ofcDNA expression vectors is allowed to infect suitable E. coli cells,followed by co-infection with a filamentous helper phage. Within thecell, trans-acting proteins encoded by the helper phage, e.g., the geneII protein of M13, recognize two separate domains positioned within thelambda arms of the vector and introduce single-stranded nicks flankingthe intiator-terminator cassette. Upon a subsequent round of DNAsynthesis, a new DNA strand is synthesized that displaces the existingnick strand liberating the initiator-terminator cassette. The displacedstrand is then circularized, packaged as filamentous phage by the helperproteins and excreted from the cell. The BK plasmid containing the cDNAis recovered by infecting an F′ strain of E. coli and plating theinfected cells on solid medium supplemented with kanamycin for theselection of pBK-containing cells.

[0403] The Renilla cDNA expression library can be screened using avariety of methods known to those of skill in the art. For example,identification of Renilla GFP may be achieved using a functionalscreening method employing blue light and observing colonies visuallyfor emission of green fluorescence or by observing light emission usingone or more bandpass filter.

[0404] 3. Cloning of Renilla reniformis Green Fluorescent Protein

[0405]Renilla reniformis GFP has 233 amino aids compared GFPs fromanimals that contain luciferase-GFP bioluminescent systems Renillamulleri, Ptilosarcus and Aequorea victoria. Other such GFPs have 238amino acids. At the amino acid level, Renilla reniformis is respectively53, 51 and 19% identical to the GFPs from these animals. The extent ofidentity of Renilla reniformis GFP to the half dozen cloned anthozoancoral GFPs, which do not contain associated luciferases, ranges from 32to 38%. The overall identity among these GFPs is surprisingly low for aprotein evolved from a common ancestor. These relationships are depictedas a phylogenetic tree (FIG. 1).

[0406] Most surprising is the finding that the Renilla reniformis GFP ismuch more closely related to Ptilosarcus GFP (77% identity) than toRenilla reniformis GFP (53%). It is unclear why the sequence relatednessbetween these 3 GFPs does not follow traditional taxonomy. Given thesequence differences at the amino acid level, coding DNA sequences aresurprisingly well conserved. Renilla reniformis GFP DNA is 56 and 59%identical to Renilla mulleri and Ptilosarcus GFP DNA.

[0407] Thus cloning Renilla reniformis GFP clone suggests why manygroups may have failed in attempts to clone this gene by traditionalmethods. An attempt to sequence the entire protein by Edman degradationwas difficult from the outset because the GFP was refractory to mostattempts at specific proteolysis. Although over 80% of the protein waseventually accurately sequenced, a 30 amino acid region (110-139 of SEQID No. 27) had not be sequenced (as well as other regions, includingamino acids 41-43, 65-71; SEQ ID No. 27). This 30 amino acid regionapparently is degraded by the proteolytic methods used into very smallfragments that are difficult to isolate and sequence; proper ordering ofsequenced fragments was also difficult.

[0408] The cloned DNA fragments can be replicated in bacterial cells,such as E. coli. A preferred DNA fragment also includes a bacterialorigin of replication, to ensure the maintenance of the DNA fragmentfrom generation to generation of the bacteria. In this way, largequantities of the DNA fragment can be produced by replication inbacteria. Preferred bacterial origins of replication include, but arenot limited to, the f1-ori and col E1 origins of replication. Preferredhosts contain chromosomal copies of DNA encoding T7 RNA polymeraseoperably linked to an inducible promoter, such as the lacUV promoter(see, U.S. Pat. No. 4,952,496). Such hosts include, but are not limitedto, lysogens E. coli strains HMS174(DE3)pLysS, BL21(DE3)pLysS,HMS174(DE3) and BL21 (DE3). Strain BL21 (DE3) is preferred. The pLysstrains provide low levels of T7 lysozyme, a natural inhibitor of T7 RNApolymerase.

[0409] For expression and for preparation of muteins, such astemperature sensitive muteins, eukaryotic cells, among them, yeastcells, such as Saccharomyces are preferred.

[0410] Nucleic acid encoding fusion proteins of the luciferases and GFPsare also provided. The resulting fusion proteins are also provided.Nucleic acids that encode luciferase and GFPs as polycistronic mRNA orunder the control of separate promoters are also provided. Methods ofuse thereof are also provided.

[0411] The GFP cloned from Renilla has spectral properties that make itextremely useful. These properties include very high quantum efficiency,high molar absorbency and efficient use with universally availablefluorescein filters (e.g., Endo GFP filter set sold by Chroma). It isknown that Renilla reniformis GFP is sixfold brighter than the wild-typeAequorea GFP on a molar basis, and three to fourfold brighter than thebrightest mutant.

[0412] The Renilla mullerei GFP encoded by the nucleic acid clonesprovided herein exhibits similar functional characteristics, and thespectra appear identical with those from native reniformis GFP. Sequencecomparison among the GFPs isolated from Aequorea victoria, Renillamullerei, and Ptilosarcus reveal that the chromophore sequences of R.mullerei and Ptilosarcus are identical, and differ from A. victoria.These sequence differences point to protein sites that can be modifiedwithout affecting the essential fluorescence properties and also providea means to identify residues that change these properties.

[0413] 4. Isolation and Identification of DNA Encoding Renilla mulleriGFP

[0414] Methods for identification and cloning of GPFs from Renilla havebeen described (see, published International PCT application No. WO99/49019, and copending allowed U.S. application Ser. No. 09/277,716).Nucleic acid encoding Renilla mulleri has been isolated. Briefly, a R.mulleri λ Uni-Zap cDNA expression plasmid library was prepared,transformed into competent E. coli cells and plated onto modifiedL-broth plates containing carbon black to absorb backgroundfluorescence. Transformants were sprayed with a solution containing IPTGto induce expression of the recombinant Renilla GFP from theheterologous cDNA. To identify GFP expressing clones, transformants wereplaced in blue light, preferably 470 to 490 nm light, and colonies thatemitted green fluorescence were isolated and grown in pure culture.

[0415] The nucleotide sequence of the cDNA insert of a green fluorescenttransformant was determined (e.g., see SEQ ID No. 15). The 1,079 cDNAinsert encodes a 238 amino acid polypeptide that is only 23.5% identicalto A. victoria GFP. The recombinant protein exhibits excitation andemission spectra similar to those reported for live Renilla species.

[0416] 5. Isolation and Identification of DNA Encoding Renilla mulleriLuciferase

[0417] The above-described R. mulleri cDNA expression library was alsoused to clone DNA encoding a R. mulleri luciferase. Single colonytransformants were grown on modified L-broth plates containing carbonblack and expression from the heterologous DNA was induced with IPTG,essentially as described above. After allowing time for expression, thetransformants were sprayed with coelenterazine and screened for thosecolonies that emit blue light. Light-emitting colonies were isolated andgrown in pure culture.

[0418] The nucleotide sequence of the cDNA insert contained in thelight-emitting transformant was determined. The 1,217 cDNA insertencodes a 311 amino acid polypeptide. The recombinant protein exhibitsexcitation and emission spectra similar to those reported for liveRenilla species.

[0419] E. Recombinant Expression of Proteins

[0420] 1. DNA Encoding Renilla Proteins

[0421] As described above, DNA encoding a Renilla GFP or Renillaluciferase can be isolated from natural sources, synthesized based onRenilla sequences provided herein or isolated as described herein.

[0422] In preferred embodiments, the DNA fragment encoding a Renilla GFPhas the sequence of amino acids set forth in SEQ ID No. 27, encoded bynucleic acid, such as that set forth SEQ ID Nos. 23-26 and 27.

[0423] A DNA molecule encoding a Renilla luciferase has the sequence ofamino acids set forth in SEQ ID No. 18. In more preferred embodiments,the DNA fragment encodes the sequence of amino acids encoded bynucleotides 31-963 of the sequence of nucleotides set forth in SEQ IDNo. 17.

[0424] 2. DNA Constructs for Recombinant Production of Renillareniformis GFP and Other Proteins

[0425] DNA is introduced into a plasmid for expression in a desiredhost. In preferred embodiments, the host is a bacterial host. Thesequences of nucleotides in the plasmids that are regulatory regions,such as promoters and operators, are operationally associated with oneanother for transcription of the sequence of nucleotides that encode aRenilla GFP or luciferase. The sequence of nucleotides encoding the FGFmutein may also include DNA encoding a secretion signal, whereby theresulting peptide is a precursor of the Renilla GFP.

[0426] In preferred embodiments the DNA plasmids also include atranscription terminator sequence. The promoter regions andtranscription terminators are each independently selected from the sameor different genes.

[0427] A wide variety of multipurpose vectors suitable for theexpression of heterologous proteins are known to those of skill in theart and are commercially available. Expression vectors containinginducible promoters or constitutive promoters that are linked toregulatory regions are preferred. Such promoters include, but are notlimited to, the T7 phage promoter and other T7-like phage promoters,such as the T3, T5 and SP6 promoters, the trp, Ipp, tet and lacpromoters, such as the lacUV5, from E. coli; the SV40 promoter; the P10or polyhedron gene promoter of baculovirus/insect cell expressionsystems, retroviral long-terminal repeats and inducible promoters fromother eukaryotic expression systems.

[0428] Particularly preferred vectors for recombinant expression ofRenilla mulleri in prokaryotic organisms are lac- and T7 promoter-basedvectors, such as the well known Bluescript vectors, which arecommercially available (Stratagene, La Jolla, Calif.).

[0429] 3. Host Organisms for Recombinant Production of Renilla Proteins

[0430] Host organisms include those organisms in which recombinantproduction of heterologous proteins have been carried out, such as, butnot limited to, bacteria (for example, E. coli yeast (for example,Saccharomyces cerevisiae and Pichia pastoris), fungi, baculovirus/insectsystems, amphibian cells, mammalian cells, plant cells and insect cells.Presently preferred host organisms are strains of bacteria or yeast.Most preferred host organisms are strains of E. coli or Saccharomycescerevisiae.

[0431] 4. Methods for Recombinant Production of Renilla Proteins

[0432] The DNA encoding a Renilla GFP or Renilla mulleri luciferase isintroduced into a plasmid in operative linkage to an appropriatepromoter for expression of polypeptides in a selected host organism. TheDNA molecule encoding the Renilla GFP or luciferase may also include aprotein secretion signal that functions in the selected host to directthe mature polypeptide into the periplasm or culture medium. Theresulting Renilla GFP or luciferase can be purified by methods routinelyused in the art, including methods described hereinafter in theExamples.

[0433] Methods of transforming suitable host cells, preferably bacterialcells, and more preferably E. coli cells, as well as methods applicablefor culturing said cells containing a gene encoding a heterologousprotein, are generally known in the art. See, for example, Sambrook etal. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.

[0434] Once the Renilla-encoding DNA molecule has been introduced intothe host cell, the desired Renilla GFP is produced by subjecting thehost cell to conditions under which the promoter is induced, whereby theoperatively linked DNA is transcribed. The cellular extracts of lysedcells containing the protein may be prepared and the resulting“clarified lysate” was employed as a source of recombinant Renilla GFPor Renilla mulleri luciferase. Alternatively, the lysate may besubjected to additional purification steps (e.g., ion exchangechromatography or immunoaffinity chromatography) to further enrich thelysate or provide a homogeneous source of the purified enzyme (see e.g.,U.S. Pat. Nos. 5,292,658 and 5,418,155).

[0435] 5. Recombinant Cells Expressing Heterologous Nucleic AcidEncoding Renilla GFP

[0436] Cells, vectors and methods are described with respect to RenillaThe same cells, vectors and methods may be used for expressingluciferases and other GFPs from species including Gaussia, Pleuromammaand Ptilosarcus.

[0437] Recombinant cells containing heterologous nucleic acid encoding aRenilla reniformis GFP are provided. In preferred embodiments, therecombinant cells express the encoded Renilla GFP which is functionaland non-toxic to the cell.

[0438] In certain embodiments, the recombinant cells may also includeheterologous nucleic acid encoding a component of abioluminescence-generating system, preferably a photoprotein orluciferase. In preferred embodiments, the nucleic acid encoding thebioluminescence-generating system component is isolated from the speciesAequorea, Vargula or Renilla. In more preferred embodiments, thebioluminescence-generating system component is a Renilla mulleriluciferase having the amino acid sequence set forth in SEQ ID No. 18.

[0439] Recombinant host cells containing heterologous nucleic acidencoding a Renilla mulleri luciferase are also provided. In preferredembodiments, the heterologous nucleic acid encodes the sequence of aminoacids as set forth in SEQ ID No. 18. In more preferred embodiments, theheterologous nucleic acid encodes the sequence of nucleotides set forthin SEQ ID No. 17.

[0440] Exemplary cells include bacteria (e.g., E. coli), plant cells,cells of mammalian origin (e.g., COS cells, mouse L cells, Chinesehamster ovary (CHO) cells, human embryonic kidney (HEK) cells, Africangreen monkey cells and other such cells known to those of skill in theart), amphibian cells (e.g., Xenopus laevis oöcytes), yeast cells (e.g.,Saccharomyces cerevisiae, Pichia pastoris), and the like. Exemplarycells for expressing injected RNA transcripts include Xenopus laevisoöcytes. Eukaryotic cells that are preferred for transfection of DNA areknown to those of skill in the art or may be empirically identified, andinclude HEK293 (which are available from ATCC under accession #CRL1573); Ltk⁻ cells (which are available from ATCC under accession#CCL1.3); COS-7 cells (which are available from ATCC under accession#CRL 1651); and DG44 cells (dhfr⁻ CHO cells; see, e.g., Urlaub et al.(1986) Cell. Molec. Genet. 12: 555). Presently preferred cells includestrains of bacteria and yeast.

[0441] The recombinant cells that contain the heterologous DNA encodingthe Renilla GFP are produced by transfection with DNA encoding a RenillaGFP or luciferase or by introduction of RNA transcripts of DNA encodinga Renilla proteins using methods well known to those of skill in theart. The DNA may be introduced as a linear DNA fragment or may beincluded in an expression vector for stable or transient expression ofthe encoding DNA. The sequences set forth herein for Renilla reniformisGFP are presently preferred (see SEQ ID Nos 23-25 and 27; see, also SEQID No. 26, which sets forth human optimized codons).

[0442] Heterologous DNA may be maintained in the cell as an episomalelement or may be integrated into chromosomal DNA of the cell. Theresulting recombinant cells may then be cultured or subcultured (orpassaged, in the case of mammalian cells) from such a culture or asubculture thereof. Also, DNA may be stably incorporated into cells ormay be transiently expressed using methods known in the art.

[0443] The recombinant cells can be used in a wide variety of cell-basedassay methods, such as those methods described for cells expressing wildtype or modified A. victoria GFPs or GFP fusion proteins (e.g., see U.S.Pat. No. 5,625,048; International patent application Publication Nos. WO95/21191; WO 96/23810; WO 96/27675; WO 97/26333; WO 97/28261; WO97/41228; and WO 98/02571).

[0444] F. Compositions and Conjugates

[0445] Compositions and conjugates and methods of use are described withreference to Renilla proteins and nucleic acids. The same compositionsand methods for preparation and use thereof are intended for use withother luciferases, such as Pleuromamma and Ptilosarcus proteins andnucleic acids.

[0446] 1. Renilla GFP Compositions

[0447] Compositions containing a Renilla GFP or GFP peptide areprovided. The compositions can take any of a number of forms, dependingon the intended method of use therefor. In certain embodiments, forexample, the compositions contain a Renilla GFP or GFP peptide,preferably Renilla mulleri GFP or Renilla reniformis GFP peptide,formulated for use in luminescent novelty items, immunoassays, FRET andFET assays. The compositions may also be used in conjunction withmulti-well assay devices containing integrated photodetectors, such asthose described herein.

[0448] Compositions that contain a Renilla mulleri GFP or GFP peptideand at least one component of a bioluminescence-generating system,preferably a luciferase, luciferin or a luciferase and a luciferin, areprovided. In preferred embodiments, the luciferase/luciferinbioluminescence-generating system is selected from those isolated from:an insect system, a coelenterate system, a ctenophore system, abacterial system, a mollusk system, a crustacea system, a fish system,an annelid system, and an earthworm system. Presently preferredbioluminescence-generating systems are those isolated from Renilla,Aequorea, and Vargula.

[0449] In more preferred embodiments, the bioluminescence-generatingsystem component is a Renilla mulleri luciferase having the amino acidsequence set forth in SEQ ID No. 18 or a Renilla reniformis luciferase.These compositions can be used in a variety of methods and systems, suchas included in conjunction with diagnostic systems for the in vivodetection of neoplastic tissues and other tissues, such as those methodsdescribed in detail below.

[0450] These methods and products include any known to those of skill inthe art in which luciferase is used, including, but not limited to U.S.application Ser. Nos. 08/757,046, 08/597,274 and 08/990,103, U.S. Pat.No. 5,625,048; International patent application Publication Nos. WO95/21191; WO 96/23810; WO 96/27675; WO 97/26333; WO 97/28261; WO97/41228; and WO 98/02571).

[0451] 2. Renilla Luciferase Compositions

[0452] DNA encoding the Renilla mulleri luciferase or Renilla reniformisluciferase is used to produce the encoded luciferase, which hasdiagnostic applications as well as use as a component of thebioluminescence generating systems as described herein, such as inbeverages, and methods of diagnosis of neoplasia and in the diagnosticchips described herein. These methods and products include any known tothose of skill in the art in which luciferase is used, including, butnot limited to, U.S. application Ser. Nos. 08/757,046, 08/597,274 and08/990,103, U.S. Pat. No. 5,625,048; International patent applicationPublication Nos. WO 95/21191; WO 96/23810; WO 96/27675; WO 97/26333; WO97/28261; WO 97/41228; and WO 98/02571).

[0453] In other embodiments, the Renilla luciferase and the remainingcomponents may be packaged as separate compositions, that, upon mixing,glow. For example, a composition containing Renilla luciferase may beprovided separately from, and use with, an a separate compositioncontaining a bioluminescence substrate and bioluminescence activator. Inanother instance, luciferase and luciferin compositions may beseparately provided and the bioluminescence activator may be addedafter, or simultaneously with, mixing of the other two compositions.

[0454] 3. Conjugates

[0455] Conjugates are provided herein for a variety of uses. Among themarer for targeting to tumors for visualization of the tumors,particularly in situ during surgery. A general description of theseconjugates and the uses thereof is described in allowed U.S. applicationSer. No. 08/908,909. In practice, prior to a surgical procedure, theconjugate is administered via any suitable route, whereby the targetingagent binds to the targeted tissue by virtue of its specific interactionwith a tissue-specific cell surface protein. During surgery the tissueis contacted, with the remaining component(s), typically by spraying thearea or local injection, and any tissue to which conjugate is bound willglow. The glow should be sufficient to see under dim light or, ifnecessary, in the dark.

[0456] The conjugates that are provided herein contain a targetingagent, such as a tissue specific or tumor specific monoclonal antibodyor fragment thereof linked either directly or via a linker to a targetedagent, a Renilla GFP, Renilla or Gaussia luciferase and otherluciferases (including photoproteins or luciferase enzymes) or aluciferin. The targeted agent may be coupled to a microcarrier. Thelinking is effected either chemically, by recombinant expression of afusion protein in instances when the targeted agent is a protein, and bycombinations of chemical and recombinant expression. The targeting agentis one that will preferentially bind to a selected tissue or cell type,such as a tumor cell surface antigen or other tissue specific antigen.

[0457] Methods for preparing conjugates are known to those of skill inthe art. For example, aequorin that is designed for conjugation andconjugates containing such aequorin have been produced (see, e.g.,International PCT application No. WO 94/18342; see, also Smith et al.(1995) in American Biotechnology Laboratory). Aequorin has beenconjugated to an antibody molecule by means of a sulfhydryl-reactingbinding agent (Stultz et al. (1992) Use of Recombinant BiotinylatedApoaequorin from Escherichia coli. Biochemistry 31, 1433-1442). Suchmethods may be adapted for use herein to produce the luciferase coupledto protein or other such molecules, which are useful as targetingagents. Vargula luciferase has also been linked to other molecules (see,e.g., Japanese application No. JP 5064583, Mar. 19, 1993). Such methodsmay be adapted for use herein to produce luciferase coupled to moleculesthat are useful as targeting agents.

[0458] The conjugates can be employed to detect the presence of orquantitate a particular antigen in a biological sample by directcorrelation to the light emitted from the bioluminescent reaction.

[0459] As an alternative, a component of the bioluminescence generatingsystem may be modified for linkage, such as by addition of amino acidresidues that are particularly suitable for linkage to the selectedsubstrate. This can be readily effected by modifying the DNA andexpressing such modified DNA to produce luciferase with additionalresidues at the N- or C-terminus.

[0460] Methods for preparing conjugates are known to those of skill inthe art. For example, aequorin that is designed for conjugation andconjugates containing such aequorin have been produced (see, e.g.,International PCT application No. WO 94/18342; see, also Smith et al.(1995) in American Biotechnology Laboratory). Aequorin has beenconjugated to an antibody molecule by means of a sulfhydryl-reactingbinding agent (Stultz et al. (1992) Use of Recombinant BiotinylatedApoaequorin from Escherichia coli. Biochemistry 31, 1433-1442). Suchmethods may be adapted for use herein to produce aequorin coupled toprotein or other such molecules, which are useful as targeting agents.Vargula luciferase has also been linked to other molecules (see, e.g.,Japanese application No. JP 5064583, Mar. 19, 1993). Such methods may beadapted for use herein to produce aequorin coupled to protein or othersuch molecules, which are useful as targeting agents. Thebioluminescence generating reactions are used with the Renillareniformis GFP provided herein.

[0461] a. Linkers

[0462] Any linker known to those of skill in the art may be used herein.Other linkers are suitable for incorporation into chemically producedconjugates. Linkers that are suitable for chemically linked conjugatesinclude disulfide bonds, thioether bonds, hindered disulfide bonds, andcovalent bonds between free reactive groups, such as amine and thiolgroups. These bonds are produced using heterobifunctional reagents toproduce reactive thiol groups on one or both of the polypeptides andthen reacting the thiol groups on one polypeptide with reactive thiolgroups or amine groups to which reactive maleimido groups or thiolgroups can be attached on the other. Other linkers include, acidcleavable linkers, such as bismaleimideothoxy propane, acidlabile-transferrin conjugates and adipic acid diihydrazide, that wouldbe cleaved in more acidic intracellular compartments; cross linkers thatare cleaved upon exposure to UV or visible light and linkers, such asthe various domains, such as C_(H)1, C_(H)2, and C_(H)3, from theconstant region of human IgG₁ (see, Batra et al. (1993) MolecularImmunol. 30:379-386). In some embodiments, several linkers may beincluded in order to take advantage of desired properties of eachlinker.

[0463] Chemical linkers and peptide linkers may be inserted bycovalently coupling the linker to the TA and the targeted agent. Theheterobifunctional agents, described below, may be used to effect suchcovalent coupling. Peptide linkers may also be linked by expressing DNAencoding the linker and TA, linker and targeted agent, or linker,targeted agent and TA as a fusion protein.

[0464] Flexible linkers and linkers that increase solubility of theconjugates are contemplated for use, either alone or with other linkersare contemplated herein.

[0465] Numerous heterobifunctional cross-linking reagents that are usedto form covalent bonds between amino groups and thiol groups and tointroduce thiol groups into proteins, are known to those of skill inthis art (see, e.g., the PIERCE CATALOG, ImmunoTechnology Catalog &Handbook, 1992-1993, which describes the preparation of and use of suchreagents and provides a commercial source for such reagents; see, also,e.g., Cumber et al. (1992) Bioconjugate Chem. 3:397-401; Thorpe et al.(1987) Cancer Res. 47:5924-5931; Gordon et al. (1987) Proc. Natl. Acad.Sci. 84:308-312; Walden et al. (1986) J. Mol. Cell Immunol. 2:191-197;Carlsson et al. (1978) Biochem. J. 173: 723-737; Mahan et al. (1987)Anal. Biochem. 162:163-170; Wawryznaczak et al. (1992) Br. J. Cancer66:361-366; Fattom et al. (1992) Infection & Immun. 60:584-589). Thesereagents may be used to form covalent bonds between the TA and targetedagent. These reagents include, but are not limited to:N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP; disulfide linker);sulfosuccinimidyl 6-(3-(2-pyridyidithio)propionamido)hexanoate(sulfo-LC-SPDP); succinimidyloxycarbonyl-α-methyl benzyl thiosulfate(SMBT, hindered disulfate linker); succinimidyl 6-(3-(2-pyridyidithio)propionamido)hexanoate (LC-SPDP); sulfosuccinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC);succinimidyl 3-(2-pyridyidithio)-butyrate (SPDB; hindered disulfide bondlinker); sulfosuccinimidyl 2-(7-azido-4-methylcoumarin-3-acetamide)ethyl-1,3′-dithiopropionate (SAED); sulfo-succinimidyl7-azido-4-methylcoumarin-3-acetate (SAMCA); sulfosuccinimidyl6-(alpha-methyl-alpha-(2-pyridyldithio)toluamido)-hexanoate(sulfo-LC-SMPT); 1,4-di-(3′-(2′-pyridyidithio)propion-amido)butane(DPDPB); 4-succinimidyloxycarbonyl-α-methyl-α-(2-pyridylthio)toluene(SMPT, hindered disulfatelinker);sulfosuccinimidyl6(α-methyl-α-(2-pyridyldithio)toluamido)hexanoate(sulfo-LC-SMPT); m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS);m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBS);N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB; thioether linker);sulfosuccinimidyl(4-iodoacetyl)amino benzoate (sulfo-SIAB);succinimidyl4(p-maleimidophenyl)butyrate (SMPB);sulfosuccinimidyl4-(p-maleimidophenyl)butyrate (sulfo-SMPB);azidobenzoyl hydrazide (ABH).

[0466] Acid cleavable linkers, photocleavable and heat sensitive linkersmay also be used, particularly where it may be necessary to cleave thetargeted agent to permit it to be more readily accessible to reaction.Acid cleavable linkers include, but are not limited to,bismaleimideothoxy propane; and adipic acid dihydrazide linkers (see,e.g., Fattom et al. (1992) Infection & Immun. 60:584-589) and acidlabile transferrin conjugates that contain a sufficient portion oftransferrin to permit entry into the intracellular transferrin cyclingpathway (see, e.g., Welhöner et al. (1991) J. Biol. Chem.266:4309-4314).

[0467] Photocleavable linkers are linkers that are cleaved upon exposureto light (see, e.g., Goldmacher et al. (1992) Bioconj. Chem. 3:104-107,which linkers are herein incorporated by reference), thereby releasingthe targeted agent upon exposure to light. Photocleavable linkers thatare cleaved upon exposure to light are known (see, e.g., Hazum et al.(1981) in Pept., Proc. Eur. Pept. Symp., 16th, Brunfeldt, K (Ed), pp.105-110, which describes the use of a nitrobenzyl group as aphotocleavable protective group for cysteine; Yen et al. (1989)Makromol. Chem 190:69-82, which describes water soluble photocleavablecopolymers, including hydroxypropylmethacrylamide copolymer, glycinecopolymer, fluorescein copolymer and methylrhodamine copolymer;Goldmacher et al. (1992) Bioconj. Chem. 3:104-107, which describes across-linker and reagent that undergoes photolytic degradation uponexposure to near UV light (350 nm); and Senter et al. (1985) Photochem.Photobiol 42:231-237, which describes nitrobenzyloxycarbonyl chloridecross linking reagents that produce photocleavable linkages), therebyreleasing the targeted agent upon exposure to light. Such linkers wouldhave particular use in treating dermatological or ophthalmic conditionsthat can be exposed to light using fiber optics. After administration ofthe conjugate, the eye or skin or other body part can be exposed tolight, resulting in release of the targeted moiety from the conjugate.Such photocleavable linkers are useful in connection with diagnosticprotocols in which it is desirable to remove the targeting agent topermit rapid clearance from the body of the animal.

[0468] b. Targeting Agents

[0469] Targeting agents include any agent that will interact with andlocalize the targeted agent cells in a tumor or specialized tissue(targeted tissue). Such agents include any agent that specificallyinteracts with a cell surface protein or receptor that is present atsufficiently higher concentrations or amounts on the targeted tissue,whereby, when contacted with an appropriate bioluminescence generatingreagent and activators produces light. These agents include, but are notlimited to, growth factors, preferentially modified to not internalize,methotrexate, and antibodies, particularly, antibodies raised againsttumor specific antigens. A plethora of tumor-specific antigens have beenidentified from a number of human neoplasms.

[0470] c. Anti-Tumor Antigen Antibodies

[0471] Polyclonal and monoclonal antibodies produced against selectedantigens. Alternatively, many such antibodies are presently available.An exemplary list of antibodies and the tumor antigen for which each hasbeen directed against is provided in U.S. application Ser. No. ______,which is incorporated by reference in its entirety. It is contemplatedthat any of the antibodies listed may be conjugated with abioluminescence generating component following the methods providedherein.

[0472] Among the preferred antibodies for use in the methods herein arethose of human origin or, more preferably, are humanized monoclonalantibodies. These are preferred for diagnosis of humans.

[0473] d. Preparation of the Conjugates

[0474] The methods for preparation of the conjugates for use in thetumor diagnostic methods can be used for preparation of the fusionproteins and conjugated proteins for use in the BRET system desribedbelow. Any method for linking proteins may be used. For example, methodsfor linking a luciferase to an antibody is described in U.S. Pat. No.5,486,455. As noted above, the targeting agent and luciferin orluciferase may be linked directly, such as through covalent bonds, i.e.,sulfhyryl bonds or other suitable bonds, or they may be linked through alinker. There may be more than one luciferase or luciferin per targetingagent, or more than one targeting agent per luciferase or luciferin.

[0475] Alternatively, an antibody, or F(Ab)₂ antigen-binding fragmentthereof or other protein targeting agent may be fused (directly or via alinking peptide) to the luciferase using recombinant DNA technology. Forexample, the DNA encoding any of the anti-tumor antibodies of Table 3may be ligated in the same translational reading frame to DNA encodingany of the above-described luciferases, e.g., SEQ ID NOs. 1-14 andinserted into an expression vector. The DNA encoding the recombinantantibody-luciferase fusion may be introduced into an appropriate host,such as bacteria or yeast, for expression.

[0476] 4. Formulation of the Compositions for Use in the DiagnosticSystems

[0477] In most embodiments, the Renilla GFPS and components of thediagnostic systems provided herein, such as Renilla luciferase, areformulated into two compositions: a first composition containing theconjugate; and a second composition containing the remaining componentsof the bioluminescence generating system. The compositions areformulated in any manner suitable for administration to an animal,particularly a mammal, and more particularly a human. Such formulationsinclude those suitable for topical, local, enteric, parenteral,intracystal, intracutaneous, intravitreal, subcutaneous, intramuscular,or intravenous administration.

[0478] For example, the conjugates, which in preferred embodiments, area targeting agent linked to a luciferase (or photoprotein) areformulated for systemic or local administration. The remainingcomponents are formulated in a separate second composition for topicalor local application. The second composition will typically contain anyother agents, such as spectral shifters that will be included in thereaction. It is preferred that the components of the second compositionare formulated in a time release manner or in some other manner thatprevents degradation and/or interaction with blood components.

[0479] a. The First Composition: Formulation of the Conjugates

[0480] As noted above, the conjugates either contain a luciferase orluciferin and a targeting agents. The preferred conjugates are formedbetween a targeting agent and a luciferase, particularly the Gaussia,Renilla mulleri or Pleuromamma luciferase. The conjugates may beformulated into pharmaceutical compositions suitable for topical, local,intravenous and systemic application. Effective concentrations of one ormore of the conjugates are mixed with a suitable pharmaceutical carrieror vehicle. The concentrations or amounts of the conjugates that areeffective requires delivery of an amount, upon administration, thatresults in a sufficient amount of targeted moiety linked to the targetedcells or tissue whereby the cells or tissue can be visualized during thesurgical procedure. Typically, the compositions are formulated forsingle dosage administration. Effective concentrations and amounts maybe determined empirically by testing the conjugates in known in vitroand in vivo systems, such as those described here; dosages for humans orother animals may then be extrapolated therefrom.

[0481] Upon mixing or addition of the conjugate(s) with the vehicle, theresulting mixture may be a solution, suspension, emulsion or the like.The form of the resulting mixture depends upon a number of factors,including the intended mode of administration and the solubility of theconjugate in the selected carrier or vehicle. The effectiveconcentration is sufficient for targeting a sufficient amount oftargeted agent to the site of interest, whereby when combined with theremaining reagents during a surgical procedure the site will glow. Suchconcentration or amount may be determined based upon in vitro and/or invivo data, such as the data from the mouse xenograft model for tumors orrabbit ophthalmic model. If necessary, pharmaceutically acceptable saltsor other derivatives of the conjugates may be prepared.

[0482] Pharmaceutical carriers or vehicles suitable for administrationof the conjugates provided herein include any such carriers known tothose skilled in the art to be suitable for the particular mode ofadministration. In addition, the conjugates may be formulated as thesole pharmaceutically ingredient in the composition or may be combinedwith other active ingredients.

[0483] The conjugates can be administered by any appropriate route, forexample, orally, parenterally, intravenously, intradermally,subcutaneously, or topically, in liquid, semi-liquid or solid form andare formulated in a manner suitable for each route of administration.Intravenous or local administration is presently preferred. Tumors andvascular proliferative disorders, will typically be visualized bysystemic, intradermal or intramuscular, modes of administration.

[0484] The conjugate is included in the pharmaceutically acceptablecarrier in an amount sufficient to produce detectable tissue and to notresult in undesirable side effects on the patient or animal. It isunderstood that number and degree of side effects depends upon thecondition for which the conjugates are administered. For example,certain toxic and undesirable side effects are tolerated when trying todiagnose life-threatening illnesses, such as tumors, that would not betolerated when diagnosing disorders of lesser consequence.

[0485] The concentration of conjugate in the composition will depend onabsorption, inactivation and excretion rates thereof, the dosageschedule, and amount administered as well as other factors known tothose of skill in the art. Typically an effective dosage should producea serum concentration of active ingredient of from about 0.1 ng/ml toabout 50-1000 μg/ml, preferably 50-100 μg/ml. The pharmaceuticalcompositions typically should provide a dosage of from about 0.01 mg toabout 100-2000 mg of conjugate, depending upon the conjugate selected,per kilogram of body weight per day. Typically, for intravenousadministration a dosage of about between 0.05 and 1 mg/kg should besufficient. Local application for, such as visualization of ophthalmictissues or local injection into joints, should provide about 1 ng up to1000 μg, preferably about 1 μg to about 100 μg, per single dosageadministration. It is understood that the amount to administer will be afunction of the conjugate selected, the indication, and possibly theside effects that will be tolerated. Dosages can be empiricallydetermined using recognized models.

[0486] The active ingredient may be administered at once, or may bedivided into a number of smaller doses to be administered at intervalsof time. It is understood that the precise dosage and duration ofadministration is a function of the disease condition being diagnosedand may be determined empirically using known testing protocols or byextrapolation from in vivo or in vitro test data. It is to be noted thatconcentrations and dosage values may also vary with the severity of thecondition to be alleviated. It is to be further understood that for anyparticular subject, specific dosage regimens should be adjusted overtime according to the individual need and the professional judgment ofthe person administering or supervising the administration of thecompositions, and that the concentration ranges set forth herein areexemplary only and are not intended to limit the scope or practice ofthe claimed compositions.

[0487] Solutions or suspensions used for parenteral, intradermal,subcutaneous, or topical application can include any of the followingcomponents: a sterile diluent, such as water for injection, salinesolution, fixed oil, polyethylene glycol, glycerine, propylene glycol orother synthetic solvent; antimicrobial agents, such as benzyl alcoholand methyl parabens; antioxidants, such as ascorbic acid and sodiumbisulfite; chelating agents, such as ethylenediaminetetraacetic acid(EDTA); buffers, such as acetates, citrates and phosphates; and agentsfor the adjustment of tonicity such as sodium chloride or dextrose.Parental preparations can be enclosed in ampules, disposable syringes ormultiple dose vials made of glass, plastic or other suitable material.

[0488] If administered intravenously, suitable carriers includephysiological saline or phosphate buffered saline (PBS), and solutionscontaining thickening and solubilizing agents, such as glucose,polyethylene glycol, and polypropylene glycol and mixtures thereof.Liposomal suspensions may also be suitable as pharmaceuticallyacceptable carriers. These may be prepared according to methods known tothose skilled in the art.

[0489] The conjugates may be prepared with carriers that protect themagainst rapid elimination from the body, such as time releaseformulations or coatings. Such carriers include controlled releaseformulations, such as, but not limited to, implants andmicroencapsulated delivery systems, and biodegradable, biocompatiblepolymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolicacid, polyorthoesters, polylacetic acid and others.

[0490] The conjugates may be formulated for local or topicalapplication, such as for topical application to the skin and mucousmembranes, such as in the eye, in the form of gels, creams, and lotionsand for application to the eye or for intracisternal or intraspinalapplication. Such solutions, particularly those intended for ophthalmicuse, may be formulated as 0.01%-10% isotonic solutions, pH about 5-7,with appropriate salts. The ophthalmic compositions may also includeadditional components, such as hyaluronic acid. The conjugates may beformulated as aerosols for topical application (see, e.g., U.S. Pat.Nos. 4,044,126, 4,414,209, and 4,364,923).

[0491] Also, the compositions for activation of the conjugate in vivoduring surgical procedures may be formulated as an aerosol. Thesecompositions contain the activators and also the remainingbioluminescence generating agent, such as luciferin, where the conjugatetargets a luciferase, or a luciferase, where the conjugate targets aluciferin, such as coelenterazine.

[0492] If oral administration is desired, the conjugate should beprovided in a composition that protects it from the acidic environmentof the stomach. For example, the composition can be formulated in anenteric coating that maintains its integrity in the stomach and releasesthe active compound in the intestine. Oral compositions will generallyinclude an inert diluent or an edible carrier and may be compressed intotablets or enclosed in gelatin capsules. For the purpose of oraladministration, the active compound or compounds can be incorporatedwith excipients and used in the form of tablets, capsules or troches.Pharmaceutically compatible binding agents and adjuvant materials can beincluded as part of the composition.

[0493] Tablets, pills, capsules, troches and the like can contain any ofthe following ingredients, or compounds of a similar nature: a binder,such as microcrystalline cellulose, gum tragacanth and gelatin; anexcipient such as starch and lactose, a disintegrating agent such as,but not limited to, alginic acid and corn starch; a lubricant such as,but not limited to, magnesium stearate; a glidant, such as, but notlimited to, colloidal silicon dioxide; a sweetening agent such assucrose or saccharin; and a flavoring agent such as peppermint, methylsalicylate, and fruit flavoring.

[0494] When the dosage unit form is a capsule, it can contain, inaddition to material of the above type, a liquid carrier such as a fattyoil. In addition, dosage unit forms can contain various other materialswhich modify the physical form of the dosage unit, for example, coatingsof sugar and other enteric agents. The conjugates can also beadministered as a component of an elixir, suspension, syrup, wafer,chewing gum or the like. A syrup may contain, in addition to the activecompounds, sucrose as a sweetening agent and certain preservatives, dyesand colorings and flavors.

[0495] The active materials can also be mixed with other activematerials that do not impair the desired action, or with materials thatsupplement the desired action, such as cis-platin for treatment oftumors.

[0496] Finally, the compounds may be packaged as articles of manufacturecontaining packaging material, one or more conjugates or compositions asprovided herein within the packaging material, and a label thatindicates the indication for which the conjugate is provided.

[0497] b. The Second Composition

[0498] The second composition will include the remaining components ofthe bioluminescence generating reaction. In preferred embodiments inwhich these components are administered systemically, the remainingcomponents include the luciferin or substrate, and optionally additionalagents, such as spectral shifters, particularly the GFPs providedherein. These components, such as the luciferin, can be formulated asdescribed above for the conjugates. In some embodiments, the luciferinor luciferase in this composition will be linked to a protein carrier orother carrier to prevent degradation or dissolution into blood cells orother cellular components.

[0499] For embodiments, in which the second composition is appliedlocally or topically, they can be formulated in a spray or aerosol orother suitable means for local or topical application.

[0500] In certain embodiments described herein, all components, exceptan activator are formulated together, such as by encapsulation in a timerelease formulation that is targeted to the tissue. Upon release thecomposition will have been localized to the desired site, and will beginto glow.

[0501] In practice, the two compositions can be administeredsimultaneously or sequentially. Typically, the first composition, whichcontains the conjugate is administered first, generally an hour or twobefore the surgery, and the second composition is then administered,either pre-operatively or during surgery.

[0502] The conjugates that are provided herein contain a targetingagent, such as a tissue specific or tumor specific monoclonal antibodyor fragment thereof linked either directly or via a linker to a targetedagent, a luciferase (including photoproteins or luciferase enzymes) or aluciferin. The targeted agent may be coupled to a microcarrier. Thelinking is effected either chemically, by recombinant expression of afusion protein in instances when the targeted agent is a protein, and bycombinations of chemical and recombinant expression. The targeting agentis one that will preferentially bind to a selected tissue or cell type,such as a tumor cell surface antigen or other tissue specific antigen.

[0503] Methods for preparing conjugates are known to those of skill inthe art. For example, aequorin that is designed for conjugation andconjugates containing such aequorin have been produced (see, e.g.,International PCT application No. WO 94/18342; see, also Smith et al.(1995) in American Biotechnology Laboratory). Aequorin has beenconjugated to an antibody molecule by means of a sulfhydryl-reactingbinding agent (Stultz et al. (1992) Use of Recombinant BiotinylatedApoaequorin from Escherichia coli (Biochemistry 31:1433-1442). Suchmethods may be adapted for use herein to produce aequorin coupled toprotein or other such molecules, which are useful as targeting agents.Vargula luciferase has also been linked to other molecules (see, e.g.,Japanese application No. JP 5064583, Mar. 19, 1993). Such methods may beadapted for use herein to produce aequorin coupled to protein or othersuch molecules, which are useful as targeting agents.

[0504] Aequorin-antibody conjugates have been employed to detect thepresence of or quantitate a particular antigen in a biological sample bydirect correlation to the light emitted from the bioluminescentreaction.

[0505] As an alternative, the Renilla GFP or Renilla mulleri or Gaussialuciferase or a component of the bioluminescence generating system maybe modified for linkage, such as by addition of amino acid residues thatare particularly suitable for linkage to the selected substrate. Thiscan be readily effected by modifying the DNA and expressing suchmodified DNA to produce luciferase with additional residues at the N- orC-terminus.

[0506] Selection of the system depends upon factors such as the desiredcolor and duration of the bioluminescence desired as well as theparticular item. Selection of the targeting agent primarily depends uponthe type and characteristics of neoplasia or tissue to be visualized andthe setting in which visualization will be performed.

[0507] The Renilla reniformis GFP is added to one or both compositionsto act as a spectral shifter.

[0508] c. Practice of the Reactions in Combination with Targeting Agents

[0509] The particular manner in which each bioluminescence system willbe combined with a selected targeting agent will be a function of theagent and the neoplasia or tissue to be visualized. In general, however,a luciferin, Renilla GFP, Renilla mulleri, Pleuromamma or Gaussialuciferase or other luciferase, of the reaction will be conjugated tothe targeting agent, administered to an animal prior to surgery. Duringthe surgery, the tissues of interest are contacted with the remainingcomponent(s) of a bioluminescence generating system. Any tissue to whichor with which the targeting agent reacts will glow.

[0510] Any color of visible light produced by a bioluminescencegenerating system is contemplated for use in the methods herein.Preferably the visible light is a combination of blue, green and/or redlight of varying intensities and wavelengths. For visualizing neoplasiaor specialty tissues through mammalian tissues or tumors deeply embeddedin tissue, longer wavelengths of visible light, ie., red and nearinfrared light, is preferred because wavelengths of near infrared lightof about 700-1300 nm are known to penetrate soft tissue and bone (e.g.,see U.S. Pat. No. 4,281,645).

[0511] In other embodiments, the conjugate can be applied to the tissuesduring surgery, such as by spraying a sterile solution over the tissues,followed by application of the remaining components. Tissues thatexpress the targeted antigen will glow.

[0512] The reagents may be provided in compositions, such assuspensions, as powders, as pastes or any in other suitable sterileform. They may be provided as sprays, aerosols, or in any suitable form.The reagents may be linked to a matrix, particularly microbeads suitablefor in vivo use and of size that they pass through capillaries.Typically all but one or more, though preferably all but one, of thecomponents necessary for the reaction will be mixed and providedtogether; reaction will be triggered contacting the mixed component(s)with the remaining component(s), such as by adding Ca²⁺, FMN withreductase, FMNH₂, ATP, air or oxygen.

[0513] In preferred embodiments the luciferase or luciferase/luciferinwill be provided in combination with the targeting agent beforeadministration to the patient. The targeting agent conjugate will thenbe contacted in vivo with the remaining components. As will becomeapparent herein, there are a multitude of ways in which each system maybe combined with a selected targeting agent.

[0514] G. Combinations

[0515]Renilla reniformis GFP can be used in combination with articles ofmanufacture to produce novelty items. The Renilla reniformis GFP can beused with a bioluminescence generating system. Such items and methodsfor preparation are described in detail in U.S. Pat. Nos. 5,876,995,6,152,358 and 6,113,886) These novelty items, which are articles ofmanufacture, are designed for entertainment, recreation and amusement,and include, but are not limited to: toys, particularly squirt guns, toycigarettes, toy “Halloween” eggs, footbags and board/card games; fingerpaints and other paints, slimy play material; textiles, particularlyclothing, such as shirts, hats and sports gear suits, threads and yarns;bubbles in bubble making toys and other toys that produce bubbles;balloons; figurines; personal items, such as bath powders, body lotions,gels, powders and creams, nail polishes, cosmetics including make-up,toothpastes and other dentifrices, soaps, body paints, and bubble bath;items such as fishing lures, inks, paper; foods, such as gelatins,icings and frostings; fish food containing luciferins and transgenicfish, particularly transgenic fish that express a luciferase; plant foodcontaining a luciferin or luciferase, preferably a luciferin for usewith transgenic plants that express luciferase; and beverages, such asbeer, wine, champagne, soft drinks, and ice cubes and ice in otherconfigurations; fountains, including liquid “fireworks” and other suchjets or sprays or aerosols of compositions that are solutions, mixtures,suspensions, powders, pastes, particles or other suitable form.

[0516] Any article of manufacture that can be combined with abioluminescence-generating system as provided herein and thereby provideentertainment, recreation and/or amusement, including use of the itemsfor recreation or to attract attention, such as for advertising goodsand/or services that are associated with a logo or trademark iscontemplated herein. Such uses may be in addition to or in conjunctionwith or in place of the ordinary or normal use of such items. As aresult of the combination, the items glow or produce, such as in thecase of squirt guns and fountains, a glowing fluid or spray of liquid orparticles.

[0517] H. Exemplary Uses of Renilla reniformis GFPs and Encoding NucleicAcid Molecules

[0518] 1. Methods for Diagnosis of Neoplasms and Other Tissues

[0519] Methods for diagnosis and visualization of tissues in vivo or insitu, preferably neoplastic tissue, using compositions containing aRenilla mulleri or Ptilosarcus GFP and/or a Renilla mulleri, Pleuromammaor Gaussia luciferase are provided. For example, the Renilla mulleri GFPprotein can be used in conjunction with diagnostic systems that rely onbioluminescence for visualizing tissues in situ, such as those describedin co-pending application Ser. No. 08/908,909. The systems areparticularly useful for visualizing and detecting neoplastic tissue andspecialty tissue, such as during non-invasive and invasive procedures.The systems include compositions containing conjugates that include atissue specific, particularly a tumor-specific, targeting agent linkedto a targeted agent, such as a Renilla reniformis GFP, a luciferase orluciferin. The systems also include a second composition that containsthe remaining components of a bioluminescence generating reaction and/orthe GFP. In some embodiments, all components, except for activators,which are provided in situ or are present in the body or tissue, areincluded in a single composition.

[0520] In particular, the diagnostic systems include two compositions. Afirst composition that contains conjugates that, in preferredembodiments, include antibodies directed against tumor antigensconjugated to a component of the bioluminescence generating reaction, aluciferase or luciferin, preferably a luciferase are provided. Incertain embodiments, conjugates containing tumor-specific targetingagents are linked to luciferases or luciferins. In other embodiments,tumor-specific targeting agents are linked to microcarriers that arecoupled with, preferably more than one of the bioluminescence generatingcomponents, preferably more than one luciferase molecule.

[0521] The second composition contains the remaining components of abioluminescence generating system, typically the luciferin or luciferasesubstrate. In some embodiments, these components, particularly theluciferin are linked to a protein, such as a serum albumin, or otherprotein carrier. The carrier and time release formulations, permitsystemically administered components to travel to the targeted tissuewithout interaction with blood cell components, such as hemoglobin thatdeactivates the luciferin or luciferase.

[0522] 2. Methods of Diagnosing Diseases

[0523] Methods for diagnosing diseases, particularly infectiousdiseases, using chip methodology, a luciferase/luciferinbioluminescence-generating system, including a Gaussia, Pleuromamma orRenilla mulleri luciferase plus a Renilla reniformis GFP, are provided.In particular, the chip includes an integrated photodetector thatdetects the photons emitted by the bioluminescence-generating system asshifted by the GFP. This chip device, which is described in copendingU.S. application Ser. No. 08/990,103, which is published asInternational PCT application No. WO 98/26277, includes an integratedphotodetector that detects the photons emitted by the bioluminescencegenerating system. The method may be practiced with any suitable chipdevice, including self-addressable and non-self addressable formats,that is modified as described herein for detection of generated photonsby the bioluminescence generating systems. The chip device providedherein is adaptable for use in an array format for the detection andidentification of infectious agents in biological specimens.

[0524] To prepare the chip, a suitable matrix for chip production isselected, the chip is fabricated by suitably derivatizing the matrix forlinkage of macromolecules, and including linkage of photodiodes,photomultipliers CCD (charge coupled device) or other suitable detector,for measuring light production; attaching an appropriate macromolecule,such as a biological molecule or anti-ligand, e.g., a receptor, such asan antibody, to the chip, preferably to an assigned location thereon.Photodiodes are presently among the preferred detectors, and specifiedherein. It is understood, however, that other suitable detectors may besubstituted therefor.

[0525] In one embodiment, the chip is made using an integrated circuitwith an array, such as an X-Y array, of photodetectors, such as thatdescribed in co-pending U.S. application Ser. No. 08/990,103 The surfaceof circuit is treated to render it inert to conditions of the diagnosticassays for which the chip is intended, and is adapted, such as byderivatization for linking molecules, such as antibodies. A selectedantibody or panel of antibodies, such as an antibody specific forparticularly bacterial antigen, is affixed to the surface of the chipabove each photodetector. After contacting the chip with a test sample,the chip is contacted with a second antibody linked to the GFP, such asthe Renilla GFP, to form a chimeric antibody—GFP fusion protein or anantibody linked to a component of a bioluminescence generating system,such as a Pleuromamma, Gaussia or R. mulleri luciferase. The antibody isspecific for the antigen. The remaining components of thebioluminescence generating reaction are added, and, if any of theantibodies linked to a component of a bioluminescence generating systemare present on the chip, light will be generated and detected by theadjacent photodetector. The photodetector is operatively linked to acomputer, which is programmed with information identifying the linkedantibodies, records the event, and thereby identifies antigens presentin the test sample.

[0526] 3. Methods for Generating Renilla mulleri Luciferase, PleuromammaLuciferase and Gaussia Luciferase Fusion Proteins with Renillareniformis GFP.

[0527] Methods for generating GFP and luciferase fusion proteins areprovided. The methods include linking DNA encoding a gene of interest,or portion thereof, to DNA encoding a Renilla reniformis GFP and aluciferase in the same translational reading frame. The encoded-proteinof interest may be linked in-frame to the amino- or carboxyl-terminus ofthe GFP or luciferase. The DNA encoding the chimeric protein is thenlinked in operable association with a promoter element of a suitableexpression vector. Alternatively, the promoter element can be obtaineddirectly from the targeted gene of interest and the promoter-containingfragment linked upstream from the GFP or luciferase coding sequence toproduce chimeric GFP proteins.

[0528] For example, a chimeric fusion containing the luciferase,preferably a Renilla luciferase, more preferably a Renilla reniformisluciferase, and Renilla reniformis GFP encoding DNA linked to theN-terminal portion of a cellulose binding domain is provided.

[0529] 4. Cell-Based Assays for Identifying Compounds

[0530] Methods for identifying compounds using recombinant cells thatexpress heterologous DNA encoding a Renilla reniformis GFP under thecontrol of a promoter element of a gene of interest are provided. Therecombinant cells can be used to identify compounds or ligands thatmodulate the level of transcription from the promoter of interest bymeasuring GFP-mediated fluorescence. Recombinant cells expressingchimeric GFPs may also be used for monitoring gene expression or proteintrafficking, or determining the cellular localization of the targetprotein by identifying localized regions of GFP-mediated fluorescencewithin the recombinant cell.

[0531] I. Kits

[0532] Kits may be prepared containing the Renilla reniformis GFP or theencoding nucleic acid moleucles (see, SEQ ID Nos. 23-26) with or withoutcomponents of a bioluminescence generating system for use in diagnosticand immunoassay methods and with the novelty items, including thosedescribed herein.

[0533] In one embodiment, the kits contain appropriate reagents and anarticle of manufacture for generating bioluminescence in combinationwith the article. These kits, for example, can be used with abubble-blowing or producing toy or with a squirt gun. These kits canalso include a reloading or charging cartridge.

[0534] In another embodiment, the kits are used for detecting andvisualizing neoplastic tissue and other tissues and include a firstcomposition that contains the Renilla reniformis GFP and a selectedluciferase, such as a Renilla mulleri, Renilla reniformis or Gaussialuciferase, and a second that contains the activating composition, whichcontains the remaining components of the bioluminescence generatingsystem and any necessary activating agents.

[0535] In other embodiments, the kits are used for detecting andidentifying diseases, particularly infectious diseases, using multi-wellassay devices and include a multi-well assay device containing aplurality of wells, each having an integrated photodetector, to which anantibody or panel of antibodies specific for one or more infectiousagents are attached, and composition containing a secondary antibody,such as an antibody specific for the infectious agent that is linked,for example, to a Renilla reniformis GFP protein, a chimericantibody-Renilla reniformis GFP fusion protein, F(Ab)₂ antibodyfragment-Renilla reniformis GFP fusion protein or to such conjugatescontaining the, for example, Gaussia or Renilla mulleri or reniformis,luciferase. A second composition containing the remaining components ofa bioluminescence generating system, such as system that emits awavelength of light within the excitation range of the GFP, such asspecies of Renilla or Aequorea, for exciting the Renilla luciferase,which produces green light that is detected by the photodetector of thedevice to indicate the presence of the agent.

[0536] In further embodiments, the kits contain the components of thediagnostic systems. The kits comprise compositions containing theconjugates, preferably Renilla GFP and a Gaussia, or Pleuromamma orRenilla mulleri luciferase and remaining bioluminescence generatingsystem components. The first composition in the kit typically containsthe targeting agent conjugated to a GFP or luciferase. The secondcomposition, contains at least the luciferin (substrate) and/orluciferase. Both compositions are formulated for systemic, local ortopical application to a mammal. In alternative embodiments, the firstcomposition contains the luciferin linked to a targeting agent, and thesecond composition contains the luciferase or the luciferase and a GFP.

[0537] In general, the packaging is non-reactive with the compositionscontained therein and where needed should exclude water and or air tothe degree those substances are required for the luminescent reaction toproceed.

[0538] Diagnostic applications may require specific packaging. Thebioluminescence generating reagents may be provided in pellets,encapsulated as micro or macro-capsules, linked to matrices, preferablybiocompatible, more preferably biodegradable matrices, and included inor on articles of manufacture, or as mixtures in chambers within anarticle of manufacture or in some other configuration. For example, acomposition containing luciferase conjugate will be provided separatelyfrom, and for use with, a separate composition containing abioluminescence substrate and bioluminescence activator.

[0539] Similarly, the Renilla reniformis GFP and selected luciferaseand/or luciferin, such as a Pleuromamma, Renilla mulleri or Gaussialuciferase or luciferin, may be provided in a composition that is amixture, suspension, solution, powder, paste or other suitablecomposition separately from or in combination with the remainingcomponents, but in the absence of an activating component. Uponcontacting the conjugate, which has been targeted to a selected tissue,with this composition the reaction commences and the tissue glows. Inpreferred embodiments, the tissue glows green emitting light near 510nm. The luciferase, GFP and bioluminescence substrate, for example, arepackaged to exclude water and/or air, the bioluminescence activator.Upon administration and release at the targeted site, the reaction withsalts or other components at the site, including air in the case ofsurgical procedures, will activate the components. In some embodiments,it is desirable to provide at least the GFPs or one component of thebioluminescence generating system linked to a matrix substrate, whichcan then be locally or systemically administered.

[0540] Suitable dispensing and packaging apparatus and matrix materialsare known to those of skill in the art, and preferably include all suchapparatus described in U.S. patent Nos. see U.S. Pat. Nos. 5,876,995,6,152,358 and 6,113,886.

[0541] J. Muteins

[0542] Muteins of the Renilla reniformis GFP are provided herein.Muteins in which conservative amino acid changes that do not alter itsability to act as an acceptor of energy generated by a Renillaluciferase/substrate reaction are provided. Also provided are muteinswith altered properties, including muteins with altered spectralproperties, muteins with altered surface properties that reducemultimerization, including dimerization.

[0543] 1. Mutation of GFP Surfaces to Disrupt Multimerization

[0544]FIG. 5 depicts the three anthozoan fluorescent protein for which acrystal structure exists, another available commercially from Clontechas dsRed (also known as drFP583, as in this alignment) (Wall et al.(2000); Nature Struct. Biol. 7:1133-1138; Yarbrough et al., (2001) Proc.Natl. Acad. Sci. U.S.A. 98: 462-467). A dark gray background depictsamino acid conservation, and a light gray background depicts sharedphysiochemical properties. These crystal structures and biochemicalcharacterization (Baird et al (2000) Proc. Natl. Acad. Sci. U.S.A. 97:11984-11989) show that dsRed exists as a obligate tetramer in vitro.Evidence also exists that dsRed multimerizes in living cells (Baird etal. (2000) Proc. Natl. Acad. Sci. U.S.A. 97: 11984-11989). Sedimentationand native gel electrophoresis studies indicate that Ptilosarcus andRenilla mullerei GFPs also form tetramers in vitro and multimerize invivo. Ptilosarcus and Renilla mullerei GFPs diverge strongly in aminoacid sequence from dsRed (39% and 38% identical, respectively).Computational polypeptide threading algorithms predict that these GFPsfold into essentially the same structure as dsRed, and also the muchmore sequence divergent Aequorea victoria GFP. Renilla reniformis GFP issimilarly related in sequence to d/Red, Ptilosarcus and Renilla mullereiGFPs (37%, 51% and 53% identical, respectively), and thus is extremelylikely to form similar multimers. Multimerization is undesirable formany applications that use GFP as the reporting moiety in chimericprotein fusion. Hence muteins in which the capacity to multimerize isreduced are provided. Thus provided are mutations Renia reniformis GFPthat disrupt the formation of GFP multimers. Such mutations may also beeffected in the Ptilosarcus and Renilla mullerei and other GFPs (seeFIG. 6).

[0545] Two interaction surfaces within the dsRed tetramer, one primarilyhydrophobic (residues marked by X) and one primarily hydrophilic(residues marked by O) have been described (see, Wall et al. (2000);Nature Struct. Biol. 7:1133-1138). In general, the correspondingresidues vary considerably between the 4 GFPs in a complex way, althoughthe physicochemical properties of the amino acids are often conserved.There are a few clusters of conserved residues, especially betweenPtilosarcus and Renilla mullerei GFPs, in keeping with their 77% overallidentity.

[0546] The scheme provided herein for disruption focuses on alteringsurface amino acid side chains so that the surfaces acquire or retain ahydrophilic character, and are also altered in their stereo-chemistry(the sizes of the side chains are altered). These GFP surface regionsroughly map to the β-sheet secondary structures that comprise the GFPβ-barrel tertiary structure. It is thus essential that the secondarystructure in any surface mutants be retained, so that the choice ofamino acid side chain substitutions is governed by this consideration.

[0547] It is also desirable to introduce mutations that alter charge.For example, such mutations are those in which R, H and K residues havebeen replaced with D, such that the hydrophobic and hydrophilic surfacesnow each contain 3 mutated residues (SEQ ID No. 33; Lys to Asp at aminoacids 108, 127 and 226, Arg to Asp at amino acids131 and 199; His to Aspat amino acid 172.

[0548] Site directed mutagenesis techniques are used to introduce aminoacid side chains that amenable to aqueous solvation, and at thatsignificantly alter surface sterochemistry. Disruption of interactingsurfaces involves loss-of-function mutagenesis. It is thus contemplatedthat altering only a few residues, perhaps even one, is sufficient.

[0549] 2. Use of Advantageous GFP Surfaces with Substituted Fluorophores

[0550] Other surfaces of GFPs may be key determinants of GFP usefulnessas reporters in living systems. A GFP surface may adventitiouslyinteract with vital cellular components, thereby contributing toGFP-induced cytoxicity. Anthozoan GFPs from bioluminescentluciferase-GFP systems serve fundamentally different biologicalfunctions than do anthozoan GFPs from coral and anemones. The Renillareniformis GFP is present in low quantity and functions as a resonanceenergy acceptor in response to a dynamic neural network that enables astartled animal to emit light flashes. A coral GFP-like protein ispresent in large quantity and apparently is used primarily as a passivepigment; it may not have evolved to dynamically interact with sensitivecellular machinery. These two classes of anthozoan fluorescent proteinsthus may have surfaces with markedly different biological properties.

[0551]FIG. 4 exemplifies the site for substitution for insertingfluorophores into the background of Ptilosarcus, Renilla mullerei andRenilla reniformis GFPs. In particular, the 20 amino acid region thatlies between two highly conserved prolines with the corresponding 20amino acid region from any other anthozoan GFP (the underlined regionscorresponds to amino acids 56-75 of SEQ ID No. 27 Renilla reniformisGFP; amino acids 59-78 of SEQ ID No. 16 Renilla mulleri GFP; and aminoacids 59-78 of SEQ ID No. 32 for Ptilosarcus GFP) is replaced ormodified. These 20 residues comprise the bulk of a polypeptide regionthat threads along the interior of the β-barrel structure that ischaracteristic of anthozoan GFPs (Wall et al. (2000) Nature Struct.Biol. 7:1133-1138; Yarbrough et al. (2001) Proc. Natl. Acad. Sci. U.S.A.98: 462-467); replacement or modification alters spectral properties.

[0552] K. Transgenic Plants and Animals

[0553] As discussed above, transgenic animals and plants that containthe nucleic acid encoding the Renilla reniformis GFP are provided.Methods for producing transgenic plants and animals that express a GFPare known (see, e.g., U.S. Pat. No. 6,020,538).

[0554] Among the transgenic plants and animals provided are those thatare novelty items, such as animals with eyes or fingernails or tusks orhair or that glows fluorescently. Transgenic food animals, such aschickens and cows and pigs are contemplated from which glowing meat andeggs (green eggs and ham) can be obtained; glowing worms can serve asfishing lures. In addition, the Renilla reniformis can serve as areporter to detect that a heterologous gene linked to the GFP gene isincorporated into the animal's genome or becomes part of the genome insome or all cells. The Renilla reniformis can similarly be used as areporter for gene therapy. The GFP can be introduced into plants to maketransgenic ornamental plants that glow, such as orchids and roses andother flowering plants. Also the GFP can be used as a marker in plants,such as by linking it to a promoter, such as Fos that responds tosecondary messages to assess signal transduction. The GFP can be linkedto adenylcyclase causing the plants to emit different spectralfrequencies as the levels of adenylcyclase change.

[0555] L. Bioluminescence Resonance Energy Transfer (BRET) System

[0556] In nature, coelenterazine-using luciferases emit broadbandblue-green light (max. ˜480 nm). Bioluminescence Resonance EnergyTransfer (BRET) is a natural phenomenon first inferred from studies ofthe hydrozoan Obelia (Morin & Hastings (1971) J. Cell Physiol.77:313-18), whereby the green bioluminescent emission observed in vivowas shown to be the result of the luciferase non-radiativelytransferring energy to an accessory green fluorescent protein (GFP).BRET was soon thereafter observed in the hydrozoan Aequorea victoria andthe anthozoan Renilla reniforms. Although energy transfer in vitrobetween purified luciferase and GFP has been demonstrated in Aequorea(Morise et al. (1974) Biochemistry 13:2656-62) and Renilla (Ward &Cormier (1976) J. Phys. Chem. 80:2289-91) systems, a key difference isthat in solution efficient radiationless energy transfer occurs only inRenilla, apparently due to the pre-association of one luciferasemolecule with one GFP homodimer (Ward & Cormier (1978) Photochem.Photobiol. 27:389-96). The blue (486 nm) luminescent emission of Renillaluciferase can be completely converted to narrow band green emission(508 nm) upon addition of proper amounts of Renilla GFP (Ward & Cormier(1976) J. Phys. Chem. 80:2289-91). GFPs accept energy from excitedstates of luciferase-substrate complexes and re-emit the light asnarrow-band green light (˜510 nm). By virtue of the non-radiative energytransfer, the quantum yield of the luciferase is increased.

[0557] Luciferases and fluorescent proteins have many well-developed andvaluable uses as protein tags and transcriptional reporters; BRETincreases the sensitivity and scope of these applications. A GFPincreases the sensitivity of the luciferase reporter by raising thequantum yield. A single luciferase fused (or chemically linked) toseveral spectrally distinct GFPs provides for the simultaneous use ofmultiple luciferase reporters, activated by addition of a singleluciferin. By preparing two fusion proteins (or chemical conjugates),each containing a GFP having a different emission wavelength fused toidentical luciferases, two or more reporters can be used with a singlesubstrate addition. Thus multiple events may be monitored or multipleassays run using a single reagent addition. Such a reporter system isself-ratioing if the distribution of luciferin is uniform orreproducible.

[0558] The ability to conveniently monitor several simultaneousmacromolecular events within a cell is a major improvement over currentbioluminescent technology. BRET also enables completely new modes ofreporting by exploiting changes in association or orientation of theluciferase and fluorescent protein. By making fusion proteins, theluciferase-GFP acceptor pair may be made to respond to changes inassociation or conformation of the fused moieties and hence serves as asensor.

[0559] Energy transfer between two fluorescent proteins (FRET) as aphysiological reporter has been reported (Miyawaki et al. (1997) Nature388:882-7), in which two different GFPs were fused to the carboxyl andamino termini of calmodulin. Changes in calcium ion concentration causeda sufficient conformational change in calmodulin to alter the level ofenergy transfer between the GFP moieties. The observed change in donoremission was ˜10% while the change in ratio was ˜1.8.

[0560]FIG. 2, reproduced from allowed copending application U.S.application Ser. No. 09/277,716, illustrates the underlying principle ofBioluminescent Resonance Energy Transfer (BRET) using GFPs andluciferase, preferably cognate luciferase, and its use as sensor: A) inisolation, a luciferase, preferably an anthozoan luciferase, emits bluelight from the coelenterazine-derived chromophore; B) in isolation, aGFP, preferably an anthozoan GFP that binds to the luciferase, that isexcited with blue-green light emits green light from its integralpeptide based fluorophore; C) when the luciferase and GFP associate as acomplex in vivo or in vitro, the luciferase non-radiatively transfersits reaction energy to the GFP fluorophore, which then emits the greenlight; D) any molecular interaction that disrupts the luciferase-GFPcomplex can be quantitatively monitored by observing the spectral shiftfrom green to blue light. Hence, the interaction or disruption thereofserves as a sensor.

[0561] The similar use of a luciferase-GFP pair in the presence ofsubstrate luciferin has important advantages. First, there is nobackground and no excitation of the acceptor from the primary excitinglight. Second, because the quantum yield of the luciferase is greatlyenhanced by nonradiative transfer to GFP, background from donor emissionis less, and the signal from the acceptor relatively greater. Third, thewavelength shift from the peak emission of luciferase (˜480 nm) to thatof the GFP (typically 508-510 nm) is large, minimizing signal overlap.All three factors combine to increase the signal-to-noise ratio. Theconcentration of the GFP acceptor can be independently ascertained byusing fluorescence.

[0562] For some applications, in vitro crosslinked or otherwise in vitromodified versions of the native proteins is contemplated. Thegenetically encoded fusion proteins have many great advantages: A) Invivo use—unlike chemistry-based luminescence or radioactivity-basedassays, fusion proteins can be genetically incorporated into livingcells or whole organisms. This greatly increases the range of possibleapplications; B) Flexible and precise modification—many differentresponse modifying elements can be reproducibly and quantitativelyincorporated into a given luciferase-GFP pair; C) Simplepurification—only one reagent would need to be purified, and itspurification could be monitored via the fluorescent protein moiety.Ligand-binding motifs can be incorporated to facilitate affinitypurification methods.

[0563] 1. Design of Sensors Based on BRET

[0564] Resonance energy transfer between two chromophores is a quantummechanical process that is exquisitely sensitive to the distance betweenthe donor and acceptor chromophores and their relative orientation inspace (Wu & Brand (1994) Anal. Biochem. 218:1-13). Efficiency of energytransfer is inversely proportional to the 6^(th) power of chromophoreseparation. In practice, the useful distance range is about 10 to 100 A,which has made resonance energy transfer a very useful technique forstudying the interactions of biological macromolecules. A variety offluorescence-based FRET biosensors have been constructed, initiallyemploying chemical fluors conjugated to proteins or membrane components,and more recently, using pairs of spectrally distinct GFP mutants(Giuliano & Taylor (1998) Trends Biotech. 16:99-146; Tsien (1998) Annu.Rev. Biochem. 67:509-44).

[0565] Although these genetically encoded GFP bioluminescence-basedbiosensors have advantages over less convenient and less precisechemical conjugate-based biosensors, all share a limitation in theirdesign: it is generally difficult to construct a biosensor in whichenergy transfer is quantitative when the chromophores are in closestapposition. It is almost impossible to arbitrarily manipulate thecomplex stereochemistry of proteins so that conjugated or intrinsicchromophores are stably positioned with minimal separation and optimalorientation. The efficiency of such biosensors are also often limited bystoichiometric imbalances between resonance energy donor and acceptor;the donor and acceptor macromolecules must be quantitatively complexedto avoid background signal emanating from uncomplexed chromophores.These limitations in general design become important when biosensorsmust be robust, convenient and cheap. Developing technologies such ashigh throughput screening for candidate drugs (using high throughputscreening (HTS) protocoals), biochips and environmental monitoringsystems would benefit greatly from modular biosensors where the signalof a rare target “hit” (e.g., complex formation between twopolypeptides) is unambiguously (statistically) distinguishable from thehuge excess of “non-hits”). Current genetically encoded FRET andbioluminescence-based biosensors display hit signals that very often areless than two-fold greater than non-hit signals, and are at best afew-fold greater (Xu et al. (1999) Proc. Natl. Acad. Sci USA 96:151-156; Miyawaki et al. (1997) Nature 388:882-7).

[0566] To solve these problems, the anthozoan GFPs, particularly theRenilla GFPs, provided herein can be used in combination with itscognate luciferases. Anthozoan luciferases-GFP complexes provide a“scaffold” upon which protein domains that confer the biologicalproperties specific to a given biosensor can be linked. Although one canconstruct many useful two component biosensors based on this scaffold,in a biosensor contemplated herein, independent protein domains thatpotentially complex with one another are respectively fused to theluciferase and the GFP.

[0567] There are many possible variations on this theme. For example, ina three component system either the luciferase or GFP can be fused to aligand-binding domain from a protein of interest or other target peptideor other moiety of interest. If the design of the fusion protein iscorrect, binding of a small molecule or protein ligand then prevents theluciferase-GFP association. The resulting combination of elements is aBRET-based biosensor; the change in spectral properties in the presenceand absence of the ligand serves as sensor. More complex protein fusionscan be designed to create two component and even single component BRETbiosensors for a multitude of uses.

[0568] The nucleic acids, and the constructs and plasmids herein, permitpreparation of a variety of configurations of fusion proteins thatinclude an anthozoan GFP, in this case Renilla reniformis, preferablywith a Renilla luciferase, more preferably with the Renilla reniformisluciferase. The nucleic acid encoding the GFP can be fused adjacent tothe nucleic acid encoding the luciferase or separated therefrom byinsertion of nucleic acid encoding, for example, a ligand-binding domainof a protein of interest. The GFP and luciferase will be bound. Uponinteraction of the ligand-binding domain with the a test compound orother moiety, the interaction of the GFP and luciferase will be alteredthereby changing the emission signal of the complex. If necessary theGFP and luciferase can be modified to fine tune the interaction to makeit more sensitive to conformational changes or to temperature or otherparameters.

[0569] 2. BRET Sensor Architectures

[0570]FIG. 3 depicts some exemplary BRET sensor architectures. The upperleft panel depicts the luciferase-GFP scaffold, the basis for therepresentative BRET sensor architectures shown here. The depicted singlepolypeptide fusion constructs place the luciferase and GFP at thepolypeptide termini, bracketing interacting protein domains of choice.The luciferase and GFP can alternatively be placed centrally within thepolypeptide, between interacting protein domains (not shown). Thisalternative arrangement is advantageous for one step proteininteraction-based cloning schemes, where cDNA fragments encodingpotential protein targets can be ligated onto one end of the construct.

[0571] Single polypeptide sensors that detect conformational changeswithin protein targets or the association-dissociation of proteintargets are well-suited for the detection of physiological signals, suchas those mediated by phosphorylation or other modification of targets,or by binding of regulatory ligands, such as hormones, to targets.Sensors based on interference are best suited to assaying the presenceof small molecules or proteins independent of any regulatory context.Quantitative assays of metabolites, such as a sugar and allergens, areamong those contemplated.

[0572] Since in vivo and in vitro luciferase-to-GFP energy transfer canbe nearly 100% efficient, binding interactions between the luciferaseand GFP must be sufficient to establish an optimal spatial relationshipbetween donor and acceptor chromophores. Optimization of theluciferase-GFP energy transfer module is important in building effectiveBRET sensors. In a single polypeptide sensor it is crucial that theluciferase-GFP interaction be weak relative to interactions betweentarget domains, thus the need for an optimized energy transfer module.In practice, either the luciferase or GFP surface can be randomlymutagenized, and an optimized luciferase-GFP scaffold then selected byscreening for either blue or green emission at two near physiologicaltemperatures (thermal endpoint-selection) using current robotic systems.This disruption of BRET is readily achievable because loss-of-functionmutants (weakened luciferase-GFP binding) are orders of magnitude morefrequent than gain-of-function mutants.

[0573] With an optimized energy transfer scaffold in hand, thermalendpoint-selection can then be used, if necessary, to optimize theinteractions between the target domains incorporated into a sensor. Thissecond round of thermal endpoint-selection may be especially importantfor the construction of interference sensors because it is essentialthat such sensors be able to “open and close” at near physiologicaltemperatures to sense interference. Thermal endpoint-selection can alsobe used to weaken the binding affinity of the analyte to theinterference sensor, making it possible to thermally wash off theanalyte and reuse the sensor, a great advantage for biochip-basedapplications.

[0574] 3. Advantages of BRET Sensors

[0575] There are many advantages to the BRET sensors provided herein.For example, BRET sensors are self-ratioing. The reporter and target areintegrated into single polypeptide. This ensures 1:1:1 stoichiometryamong luciferase, GFP and target (or a 1:N:1 stoichiometry if more thanone, typically a homodimer, GFP can be bound to a luciferase). GFPfluorescence allows absolute quantitation of sensor. The null stategives signal that verifies sensor functionality. Quantifiable null statefacilitates disruption-of-BRET sensors (DBRET). BRET sensors have bettersignal-to-noise ratio than GFP FRET sensors because there is no cellularautofluorescence, no excitation of the acceptor from the primaryexciting light, the quantum yield of luciferase greatly enhanced bynon-radiative energy transfer to GFP, and there is minimal signaloverlap between emission of the luciferase and emission of the GFP.Also, anthozoan GFPs have 6-fold higher extinction coefficients thanAequorea GFP.

[0576] The BRET sensors can for used for hit identification anddownstream evaluation in in vitro screening assays in in vitro or invivo or in situ, including in cultured cells and tissues and animals.The BRET sensors can be created by thermal endpoint-selection, which issuited to DBRET (Disruption-of-BRET) and reduces need for knowledge oftarget 3D structure and functional dynamics. Existing screening roboticsto optimize biosensors. BRET sensors benefit from vast genetic diversityanthozoans have evolved efficient luciferase-GFP energy transfer systemsand the components can be mixed and matched. Highly efficientheterologous luciferases may be substituted for less active luciferases.For example, a copepod luciferase active site can be fused to ananthozoan luciferase GFP-binding domain. There are many diversecoelenterazine-using luciferases.

[0577] BRET sensors are modular so that an optimized sensor scaffold maybe used with different targets. Also the BRET acceptor may be varied togive shifted emissions, facilitating multiple simultaneous readouts. Theanthozoan GFPs can be mutated, GPFs or other proteins can be modifiedwith different chemical fluors, high throughput screening (HTS)fluor-modified FRET acceptors can be adapted, the BRET donor(luciferase) may be varied, such as by using an Aequorin (Ca++activated) photoprotein, or a firefly luciferse (requires ATP and afirefly luciferin) to give conditional activation. The sensor scaffoldcan be incorporated into a variety of immobilization motifs, includingfree format plates, which can reduce reagent volumes, reusablemicrotiter plates, miniature columns and biochips. Finally, BRET sensorsare inexpensive and reproducible reagents because they can be producedby standardized protein production and can incorporate purificationtags. Genetically encoded reporters more reproducible than chemicallymodified reporters. Linear translation of BRET modules ensures sensorintegrity.

[0578] The following example is included for illustrative purposes onlyand is not intended to limit the scope of the invention.

EXAMPLE

[0579] Specimens of the sea pansy Renilla reniformis were collected frominshore waters off the coast of Georgia. To prepare the sea pansies forisolation of mRNA, about 25 or so at time were placed on a large bed ofdry ice. They were flipped with a spatula to flip them over to preventthem from freezing. Oddly, the entire animal illuminated when it came incontact with the dry ice. The brightest and greenest were culled, placedin a bag and back into sea water at about 65-70° C. for two hours. Thisprocess of dry ice, culling and sea water treatment was repeated threetime over a 6 hour period. In addition, the process was performed atnight. After they were exhausted were the last chilling, the culledanimals were frozen solid. A cDNA library was prepared from the frozenanimals.

[0580] The animals that were selected this way were frozen in liquidnitrogen, and shipped to Stratagene, Inc. (La Jolla, Calif.). acommercial vendor whose business includes the construction of customcDNA libraries under contract to prepare the library. PurifiedpolyA-mRNA was prepared and a cDNA synthesis reaction was performed,appending a 3′ XhoI site and a 5′ EcoRI restriction site to the cDNA.The cDNA was inserted by ligation between the EcoRI and XhoI sites ofthe Uni-ZAP Lambda phage cDNA cloning vector.

[0581] The resulting unamplified library contained approximately 1.6×10⁸primary plaques, which after amplification gave a titer of 3.5-7.5 pfb(plaque forming units)/ml. Insert sizes ranged from 0.9 to 3.0 kb, withan average size around 1.5 kb. Two mass excisions were performed to givepBluescript phagemid containing the cDNA inserts; each excision fromabout 8×10 plaques gave rise to about 4.8×10⁹ cfu (colony formingunits)/ml. Phagemids were transfected into the SOLR strain of E. coli.

[0582] Screening was performed by plating (using an artist's airbrush)approximately 200,000 colonies to each of 40 cafeteria trays containingLB agar medium incorporating 0.4% carbon black to absorb backgroundfluorescence. After 24 hours growth at 30° C. in a humidified incubator,GFP expressing colonies were identified by illuminating the plates usinga 250 Watt quartz halogen fiber optics light (Cuda Products Corp) withan EGFP bandpass excitation filter (Chroma), and viewing coloniesthrough a GFP bandpass emission filter. Approximately 10 fluorescentcolonies were picked, DNA isolated from minipreps, and the DNAtransformed into the XL-10 Gold E. coli strain (Stratagene). Analysis byrestriction digestion resolved three distinguishable sizes of insert.DNA was prepared from a clone of each size class and sent to SeqWrightLLB (Houston, Tex.) for sequencing. Sequencing data were reported toProlume on 1-25-99.

[0583] Three independent cDNA clones of Renilla reniformis GFP wereisolated (SEQ ID Nos 23-25). Each cDNA is full length as judged byidentical 5′ termini and each encodes an identical protein of 233 aminoacids (see SEQ ID No. 27). Compared to the primary clone (Clone 1), thecoding sequence of Clone 2 differs by 4 silent mutations. Clones 2 and 3also contain small differences in the 5′ and 3′ untranslated regions ofthe cDNA. This nucleic acid has been inserted into expression vector,and the encoded protein produced.

[0584] Since modifications may be apparent to those of skill in the art,it is intended that the invention be limited only by the appendedclaims.

1 33 1 1196 DNA Renilla reniformis CDS (1)...(942) Renilla reniformasluciferase 1 agc tta aag atg act tcg aaa gtt tat gat cca gaa caa agg aaacgg 48 Ser Leu Lys Met Thr Ser Lys Val Tyr Asp Pro Glu Gln Arg Lys Arg 15 10 15 atg ata act ggt ccg cag tgg tgg gcc aga tgt aaa caa atg aat gtt96 Met Ile Thr Gly Pro Gln Trp Trp Ala Arg Cys Lys Gln Met Asn Val 20 2530 ctt gat tca ttt att aat tat tat gat tca gaa aaa cat gca gaa aat 144Leu Asp Ser Phe Ile Asn Tyr Tyr Asp Ser Glu Lys His Ala Glu Asn 35 40 45gct gtt att ttt tta cat ggt aac gcg gcc tct tct tat tta tgg cga 192 AlaVal Ile Phe Leu His Gly Asn Ala Ala Ser Ser Tyr Leu Trp Arg 50 55 60 catgtt gtg cca cat att gag cca gta gcg cgg tgt att ata cca gat 240 His ValVal Pro His Ile Glu Pro Val Ala Arg Cys Ile Ile Pro Asp 65 70 75 80 cttatt ggt atg ggc aaa tca ggc aaa tct ggt aat ggt tct tat agg 288 Leu IleGly Met Gly Lys Ser Gly Lys Ser Gly Asn Gly Ser Tyr Arg 85 90 95 tta cttgat cat tac aaa tat ctt act gca tgg ttg aac ttc tta att 336 Leu Leu AspHis Tyr Lys Tyr Leu Thr Ala Trp Leu Asn Phe Leu Ile 100 105 110 tac caaaga aga tca ttt ttt gtc ggc cat gat tgg ggt gct tgt ttg 384 Tyr Gln ArgArg Ser Phe Phe Val Gly His Asp Trp Gly Ala Cys Leu 115 120 125 gca tttcat tat agc tat gag cat caa gat aag atc aaa gca ata gtt 432 Ala Phe HisTyr Ser Tyr Glu His Gln Asp Lys Ile Lys Ala Ile Val 130 135 140 cac gctgaa agt gta gta gat gtg att gaa tca tgg gat gaa tgg cct 480 His Ala GluSer Val Val Asp Val Ile Glu Ser Trp Asp Glu Trp Pro 145 150 155 160 gatatt gaa gaa gat att gcg ttg atc aaa tct gaa gaa gga gaa aaa 528 Asp IleGlu Glu Asp Ile Ala Leu Ile Lys Ser Glu Glu Gly Glu Lys 165 170 175 atggtt ttg gag aat aac ttc ttc gtg gaa acc atg ttg cca tca aaa 576 Met ValLeu Glu Asn Asn Phe Phe Val Glu Thr Met Leu Pro Ser Lys 180 185 190 atcatg aga aag tta gaa cca gaa gaa ttt gca gca tat ctt gaa cca 624 Ile MetArg Lys Leu Glu Pro Glu Glu Phe Ala Ala Tyr Leu Glu Pro 195 200 205 ttcaaa gag aaa ggt gaa gtt cgt cgt cca aca tta tca tgg cct cgt 672 Phe LysGlu Lys Gly Glu Val Arg Arg Pro Thr Leu Ser Trp Pro Arg 210 215 220 gaaatc ccg tta gta aaa ggt ggt aaa cct gac gtt gta caa att gtt 720 Glu IlePro Leu Val Lys Gly Gly Lys Pro Asp Val Val Gln Ile Val 225 230 235 240agg aat tat aat gct tat cta cgt gca agt gat gat tta cca aaa atg 768 ArgAsn Tyr Asn Ala Tyr Leu Arg Ala Ser Asp Asp Leu Pro Lys Met 245 250 255ttt att gaa tcg gat cca gga ttc ttt tcc aat gct att gtt gaa ggc 816 PheIle Glu Ser Asp Pro Gly Phe Phe Ser Asn Ala Ile Val Glu Gly 260 265 270gcc aag aag ttt cct aat act gaa ttt gtc aaa gta aaa ggt ctt cat 864 AlaLys Lys Phe Pro Asn Thr Glu Phe Val Lys Val Lys Gly Leu His 275 280 285ttt tcg caa gaa gat gca cct gat gaa atg gga aaa tat atc aaa tcg 912 PheSer Gln Glu Asp Ala Pro Asp Glu Met Gly Lys Tyr Ile Lys Ser 290 295 300ttc gtt gag cga gtt ctc aaa aat gaa caa taattacttt ggttttttat 962 PheVal Glu Arg Val Leu Lys Asn Glu Gln 305 310 ttacattttt cccgggtttaataatataaa tgtcattttc aacaatttta ttttaactga 1022 atatttcaca gggaacattcatatatgttg attaatttag ctcgaacttt actctgtcat 1082 atcattttgg aatattacctctttcaatga aactttataa acagtggttc aattaattaa 1142 tatatattat aattacatttgttatgtaat aaactcggtt ttattataaa aaaa 1196 2 1822 DNA Cypridinahilagendorfii luciferase CDS (1)...(1665) Cypridina hilgendorfiiluciferase 2 atg aag cta ata att ctg tct att ata ttg gcc tac tgt gtc acagtc 48 Met Lys Leu Ile Ile Leu Ser Ile Ile Leu Ala Tyr Cys Val Thr Val 15 10 15 aac tgc cag gat gca tgt cct gta gaa gct gaa gca ccg tca agt aca96 Asn Cys Gln Asp Ala Cys Pro Val Glu Ala Glu Ala Pro Ser Ser Thr 20 2530 cca aca gtc cca aca tct tgt gaa gct aaa gaa gga gaa tgt atc gat 144Pro Thr Val Pro Thr Ser Cys Glu Ala Lys Glu Gly Glu Cys Ile Asp 35 40 45acc aga tgc gca aca tgt aaa cga gac ata cta tca gac gga ctg tgt 192 ThrArg Cys Ala Thr Cys Lys Arg Asp Ile Leu Ser Asp Gly Leu Cys 50 55 60 gaaaat aaa cca ggg aag aca tgc tgt aga atg tgc cag tat gta att 240 Glu AsnLys Pro Gly Lys Thr Cys Cys Arg Met Cys Gln Tyr Val Ile 65 70 75 80 gaatcc aga gta gaa gct gct gga tat ttt aga acg ttt tac gcc aaa 288 Glu SerArg Val Glu Ala Ala Gly Tyr Phe Arg Thr Phe Tyr Ala Lys 85 90 95 aga tttaat ttt cag gaa cct ggt aaa tat gtg ctg gct cga gga acc 336 Arg Phe AsnPhe Gln Glu Pro Gly Lys Tyr Val Leu Ala Arg Gly Thr 100 105 110 aag ggtggc gac tgg tct gta acc ctc acc atg gag aat cta gat gga 384 Lys Gly GlyAsp Trp Ser Val Thr Leu Thr Met Glu Asn Leu Asp Gly 115 120 125 cag aaggga gct gta ctg act aag aca aca ctg gag gta gta gga gac 432 Gln Lys GlyAla Val Leu Thr Lys Thr Thr Leu Glu Val Val Gly Asp 130 135 140 gta atagac att act caa gct act gca gat cct atc aca gtt aac gga 480 Val Ile AspIle Thr Gln Ala Thr Ala Asp Pro Ile Thr Val Asn Gly 145 150 155 160 ggagct gac cca gtt atc gct aac ccg ttc aca att ggt gag gtg acc 528 Gly AlaAsp Pro Val Ile Ala Asn Pro Phe Thr Ile Gly Glu Val Thr 165 170 175 attgct gtt gtc gaa ata ccc ggc ttc aat att aca gtc atc gaa ttc 576 Ile AlaVal Val Glu Ile Pro Gly Phe Asn Ile Thr Val Ile Glu Phe 180 185 190 tttaaa cta atc gtg ata gat att ctg gga gga aga tct gtg aga att 624 Phe LysLeu Ile Val Ile Asp Ile Leu Gly Gly Arg Ser Val Arg Ile 195 200 205 gctcca gac aca gca aac aaa gga ctg ata tct ggt atc tgt ggt aat 672 Ala ProAsp Thr Ala Asn Lys Gly Leu Ile Ser Gly Ile Cys Gly Asn 210 215 220 ctggag atg aat gac gct gat gac ttt act aca gac gca gat cag ctg 720 Leu GluMet Asn Asp Ala Asp Asp Phe Thr Thr Asp Ala Asp Gln Leu 225 230 235 240gcg atc caa ccc aac ata aac aaa gag ttc gac ggc tgc cca ttc tac 768 AlaIle Gln Pro Asn Ile Asn Lys Glu Phe Asp Gly Cys Pro Phe Tyr 245 250 255ggg aat cct tct gat atc gaa tac tgc aaa ggt ctc atg gag cca tac 816 GlyAsn Pro Ser Asp Ile Glu Tyr Cys Lys Gly Leu Met Glu Pro Tyr 260 265 270aga gct gta tgt cgt aac aat atc aac ttc tac tat tac act ctg tcc 864 ArgAla Val Cys Arg Asn Asn Ile Asn Phe Tyr Tyr Tyr Thr Leu Ser 275 280 285tgc gcc ttc gct tac tgt atg gga gga gaa gaa aga gct aaa cac gtc 912 CysAla Phe Ala Tyr Cys Met Gly Gly Glu Glu Arg Ala Lys His Val 290 295 300ctt ttc gac tat gtt gag aca tgc gct gca ccg gaa acg aga gga acg 960 LeuPhe Asp Tyr Val Glu Thr Cys Ala Ala Pro Glu Thr Arg Gly Thr 305 310 315320 tgt gtt tta tca gga cat act ttc tat gac aca ttc gac aaa gcc aga 1008Cys Val Leu Ser Gly His Thr Phe Tyr Asp Thr Phe Asp Lys Ala Arg 325 330335 tat caa ttc cag ggc cca tgc aaa gag ctt ctg atg gcc gca gac tgt 1056Tyr Gln Phe Gln Gly Pro Cys Lys Glu Leu Leu Met Ala Ala Asp Cys 340 345350 tac tgg aac aca tgg gat gta aag gtt tca cat aga gat gtt gag tca 1104Tyr Trp Asn Thr Trp Asp Val Lys Val Ser His Arg Asp Val Glu Ser 355 360365 tac act gag gta gag aaa gta aca atc agg aaa cag tca act gta gta 1152Tyr Thr Glu Val Glu Lys Val Thr Ile Arg Lys Gln Ser Thr Val Val 370 375380 gat ttg att gtg gat ggc aag cag gtc aag gtt gga gga gtg gat gta 1200Asp Leu Ile Val Asp Gly Lys Gln Val Lys Val Gly Gly Val Asp Val 385 390395 400 tct atc ccg tac agt tct gag aac aca tcc ata tac tgg cag gat gga1248 Ser Ile Pro Tyr Ser Ser Glu Asn Thr Ser Ile Tyr Trp Gln Asp Gly 405410 415 gac atc ctg acg acg gcc atc cta cct gaa gct ctt gtc gtt aag ttc1296 Asp Ile Leu Thr Thr Ala Ile Leu Pro Glu Ala Leu Val Val Lys Phe 420425 430 aac ttt aag cag ctc ctt gta gtt cat atc aga gat cca ttc gat gga1344 Asn Phe Lys Gln Leu Leu Val Val His Ile Arg Asp Pro Phe Asp Gly 435440 445 aag aca tgc ggc ata tgt ggt aac tat aat caa gat tca act gat gat1392 Lys Thr Cys Gly Ile Cys Gly Asn Tyr Asn Gln Asp Ser Thr Asp Asp 450455 460 ttc ttt gac gca gaa gga gca tgc gct ctg acc ccc aat ccc cca gga1440 Phe Phe Asp Ala Glu Gly Ala Cys Ala Leu Thr Pro Asn Pro Pro Gly 465470 475 480 tgt aca gag gag cag aaa cca gaa gct gag cga ctc tgc aat agtcta 1488 Cys Thr Glu Glu Gln Lys Pro Glu Ala Glu Arg Leu Cys Asn Ser Leu485 490 495 ttt gat agt tct atc gac gag aaa tgt aat gtc tgc tac aag cctgac 1536 Phe Asp Ser Ser Ile Asp Glu Lys Cys Asn Val Cys Tyr Lys Pro Asp500 505 510 cgt att gca cga tgt atg tac gag tat tgc ctg agg gga cag caagga 1584 Arg Ile Ala Arg Cys Met Tyr Glu Tyr Cys Leu Arg Gly Gln Gln Gly515 520 525 ttc tgt gac cat gct tgg gag ttc aaa aaa gaa tgc tac ata aagcat 1632 Phe Cys Asp His Ala Trp Glu Phe Lys Lys Glu Cys Tyr Ile Lys His530 535 540 gga gac act cta gaa gta cca cct gaa tgc caa taaatgaacaaagatacaga 1685 Gly Asp Thr Leu Glu Val Pro Pro Glu Cys Gln 545 550 555agctaagact actacagcag aagataaaag agaagctgta gttcttcaaa aacagtatat 1745tttgatgtac tcattgttta cttacataaa aataaattgt tattatcata acgtaaagaa 1805aaaaaaaaaa aaaaaaa 1822 3 1644 DNA Luciola cruciata CDS (1)...(1644)Luciola cruciata luciferase 3 atg gaa aac atg gaa aac gat gaa aat attgta gtt gga cct aaa ccg 48 Met Glu Asn Met Glu Asn Asp Glu Asn Ile ValVal Gly Pro Lys Pro 1 5 10 15 ttt tac cct atc gaa gag gga tct gct ggaaca caa tta cgc aaa tac 96 Phe Tyr Pro Ile Glu Glu Gly Ser Ala Gly ThrGln Leu Arg Lys Tyr 20 25 30 atg gag cga tat gca aaa ctt ggc gca att gctttt aca aat gca gtt 144 Met Glu Arg Tyr Ala Lys Leu Gly Ala Ile Ala PheThr Asn Ala Val 35 40 45 act ggt gtt gat tat tct tac gcc gaa tac ttg gagaaa tca tgt tgt 192 Thr Gly Val Asp Tyr Ser Tyr Ala Glu Tyr Leu Glu LysSer Cys Cys 50 55 60 cta gga aaa gct ttg caa aat tat ggt ttg gtt gtt gatggc aga att 240 Leu Gly Lys Ala Leu Gln Asn Tyr Gly Leu Val Val Asp GlyArg Ile 65 70 75 80 gcg tta tgc agt gaa aac tgt gaa gaa ttt ttt att cctgta ata gcc 288 Ala Leu Cys Ser Glu Asn Cys Glu Glu Phe Phe Ile Pro ValIle Ala 85 90 95 gga ctg ttt ata ggt gta ggt gtt gca ccc act aat gag atttac act 336 Gly Leu Phe Ile Gly Val Gly Val Ala Pro Thr Asn Glu Ile TyrThr 100 105 110 tta cgt gaa ctg gtt cac agt tta ggt atc tct aaa cca acaatt gta 384 Leu Arg Glu Leu Val His Ser Leu Gly Ile Ser Lys Pro Thr IleVal 115 120 125 ttt agt tct aaa aaa ggc tta gat aaa gtt ata aca gta cagaaa aca 432 Phe Ser Ser Lys Lys Gly Leu Asp Lys Val Ile Thr Val Gln LysThr 130 135 140 gta act act att aaa acc att gtt ata cta gat agc aaa gttgat tat 480 Val Thr Thr Ile Lys Thr Ile Val Ile Leu Asp Ser Lys Val AspTyr 145 150 155 160 cga gga tat caa tgt ctg gac acc ttt ata aaa aga aacact cca cca 528 Arg Gly Tyr Gln Cys Leu Asp Thr Phe Ile Lys Arg Asn ThrPro Pro 165 170 175 ggt ttt caa gca tcc agt ttc aaa act gtg gaa gtt gaccgt aaa gaa 576 Gly Phe Gln Ala Ser Ser Phe Lys Thr Val Glu Val Asp ArgLys Glu 180 185 190 caa gtt gct ctt ata atg aac tct tcg ggt tct acc ggtttg cca aaa 624 Gln Val Ala Leu Ile Met Asn Ser Ser Gly Ser Thr Gly LeuPro Lys 195 200 205 ggc gta caa ctt act cac gaa aat aca gtc act aga ttttct cat gct 672 Gly Val Gln Leu Thr His Glu Asn Thr Val Thr Arg Phe SerHis Ala 210 215 220 aga gat ccg att tat ggt aac caa gtt tca cca ggc accgct gtt tta 720 Arg Asp Pro Ile Tyr Gly Asn Gln Val Ser Pro Gly Thr AlaVal Leu 225 230 235 240 act gtc gtt cca ttc cat cat ggt ttt ggt atg ttcact act cta ggg 768 Thr Val Val Pro Phe His His Gly Phe Gly Met Phe ThrThr Leu Gly 245 250 255 tat tta att tgt ggt ttt cgt gtt gta atg tta acaaaa ttc gat gaa 816 Tyr Leu Ile Cys Gly Phe Arg Val Val Met Leu Thr LysPhe Asp Glu 260 265 270 gaa aca ttt tta aaa act cta caa gat tat aaa tgtaca agt gtt att 864 Glu Thr Phe Leu Lys Thr Leu Gln Asp Tyr Lys Cys ThrSer Val Ile 275 280 285 ctt gta ccg acc ttg ttt gca att ctc aac aaa agtgaa tta ctc aat 912 Leu Val Pro Thr Leu Phe Ala Ile Leu Asn Lys Ser GluLeu Leu Asn 290 295 300 aaa tac gat ttg tca aat tta gtt gag att gca tctggc gga gca cct 960 Lys Tyr Asp Leu Ser Asn Leu Val Glu Ile Ala Ser GlyGly Ala Pro 305 310 315 320 tta tca aaa gaa gtt ggt gaa gct gtt gct agacgc ttt aat ctt ccc 1008 Leu Ser Lys Glu Val Gly Glu Ala Val Ala Arg ArgPhe Asn Leu Pro 325 330 335 ggt gtt cgt caa ggt tat ggt tta aca gaa acaaca tct gcc att att 1056 Gly Val Arg Gln Gly Tyr Gly Leu Thr Glu Thr ThrSer Ala Ile Ile 340 345 350 att aca cca gaa gga gac gat aaa cca gga gcttct gga aaa gtc gtg 1104 Ile Thr Pro Glu Gly Asp Asp Lys Pro Gly Ala SerGly Lys Val Val 355 360 365 ccg ttg ttt aaa gca aaa gtt att gat ctt gatacc aaa aaa tct tta 1152 Pro Leu Phe Lys Ala Lys Val Ile Asp Leu Asp ThrLys Lys Ser Leu 370 375 380 ggt cct aac aga cgt gga gaa gtt tgt gtt aaagga cct atg ctt atg 1200 Gly Pro Asn Arg Arg Gly Glu Val Cys Val Lys GlyPro Met Leu Met 385 390 395 400 aaa ggt tat gta aat aat cca gaa gca acaaaa gaa ctt att gac gaa 1248 Lys Gly Tyr Val Asn Asn Pro Glu Ala Thr LysGlu Leu Ile Asp Glu 405 410 415 gaa ggt tgg ctg cac acc gga gat att ggatat tat gat gaa gaa aaa 1296 Glu Gly Trp Leu His Thr Gly Asp Ile Gly TyrTyr Asp Glu Glu Lys 420 425 430 cat ttc ttt att gtc gat cgt ttg aag tcttta atc aaa tac aaa gga 1344 His Phe Phe Ile Val Asp Arg Leu Lys Ser LeuIle Lys Tyr Lys Gly 435 440 445 tac caa gta cca cct gcc gaa tta gaa tccgtt ctt ttg caa cat cca 1392 Tyr Gln Val Pro Pro Ala Glu Leu Glu Ser ValLeu Leu Gln His Pro 450 455 460 tct atc ttt gat gct ggt gtt gcc ggc gttcct gat cct gta gct ggc 1440 Ser Ile Phe Asp Ala Gly Val Ala Gly Val ProAsp Pro Val Ala Gly 465 470 475 480 gag ctt cca gga gcc gtt gtt gta ctggaa agc gga aaa aat atg acc 1488 Glu Leu Pro Gly Ala Val Val Val Leu GluSer Gly Lys Asn Met Thr 485 490 495 gaa aaa gaa gta atg gat tat gtt gcaagt caa gtt tca aat gca aaa 1536 Glu Lys Glu Val Met Asp Tyr Val Ala SerGln Val Ser Asn Ala Lys 500 505 510 cgt tta cgt ggt ggt gtt cgt ttt gtggat gaa gta cct aaa ggt ctt 1584 Arg Leu Arg Gly Gly Val Arg Phe Val AspGlu Val Pro Lys Gly Leu 515 520 525 act gga aaa att gac ggc aga gca attaga gaa atc ctt aag aaa cca 1632 Thr Gly Lys Ile Asp Gly Arg Ala Ile ArgGlu Ile Leu Lys Lys Pro 530 535 540 gtt gct aag atg 1644 Val Ala Lys Met545 4 1820 DNA Vargula (cypridina) CDS (1)...(1665) Vargula (cypridina)luciferase 4 atg aag ata ata att ctg tct gtt ata ttg gcc tac tgt gtc accgac 48 Met Lys Ile Ile Ile Leu Ser Val Ile Leu Ala Tyr Cys Val Thr Asp 15 10 15 aac tgt caa gat gca tgt cct gta gaa gcg gaa ccg cca tca agt aca96 Asn Cys Gln Asp Ala Cys Pro Val Glu Ala Glu Pro Pro Ser Ser Thr 20 2530 cca aca gtt cca act tct tgt gaa gct aaa gaa gga gaa tgt ata gat 144Pro Thr Val Pro Thr Ser Cys Glu Ala Lys Glu Gly Glu Cys Ile Asp 35 40 45acc aga tgc gca aca tgt aaa cga gat ata cta tca gat gga ctg tgt 192 ThrArg Cys Ala Thr Cys Lys Arg Asp Ile Leu Ser Asp Gly Leu Cys 50 55 60 gaaaat aaa cca ggg aag aca tgc tgt aga atg tgc cag tat gtg att 240 Glu AsnLys Pro Gly Lys Thr Cys Cys Arg Met Cys Gln Tyr Val Ile 65 70 75 80 gaatgc aga gta gaa gca gct ggt tat ttt aga acg ttt tac ggc aaa 288 Glu CysArg Val Glu Ala Ala Gly Tyr Phe Arg Thr Phe Tyr Gly Lys 85 90 95 aga tttaat ttt cag gaa cct ggt aaa tat gtg ctg gct agg gga acc 336 Arg Phe AsnPhe Gln Glu Pro Gly Lys Tyr Val Leu Ala Arg Gly Thr 100 105 110 aag ggtggc gat tgg tct gta acc ctc acc atg gag aat cta gat gga 384 Lys Gly GlyAsp Trp Ser Val Thr Leu Thr Met Glu Asn Leu Asp Gly 115 120 125 cag aaggga gct gtg ctg act aag aca aca ctg gag gtt gca gga gac 432 Gln Lys GlyAla Val Leu Thr Lys Thr Thr Leu Glu Val Ala Gly Asp 130 135 140 gta atagac att act caa gct act gca gat cct atc aca gtt aac gga 480 Val Ile AspIle Thr Gln Ala Thr Ala Asp Pro Ile Thr Val Asn Gly 145 150 155 160 ggagct gac cca gtt atc gct aac ccg ttc aca att ggt gag gtg acc 528 Gly AlaAsp Pro Val Ile Ala Asn Pro Phe Thr Ile Gly Glu Val Thr 165 170 175 attgct gtt gtt gaa ata ccg ggc ttc aat atc aca gtc atc gaa ttc 576 Ile AlaVal Val Glu Ile Pro Gly Phe Asn Ile Thr Val Ile Glu Phe 180 185 190 tttaaa cta atc gtg att gat att ctg gga gga aga tct gtc aga att 624 Phe LysLeu Ile Val Ile Asp Ile Leu Gly Gly Arg Ser Val Arg Ile 195 200 205 gctcca gac aca gca aac aaa gga ctg ata tct ggt atc tgt ggt aat 672 Ala ProAsp Thr Ala Asn Lys Gly Leu Ile Ser Gly Ile Cys Gly Asn 210 215 220 ctggag atg aat gac gct gat gac ttt act aca gat gca gat cag ctg 720 Leu GluMet Asn Asp Ala Asp Asp Phe Thr Thr Asp Ala Asp Gln Leu 225 230 235 240gcg atc caa ccc aac ata aac aaa gag ttc gac ggc tgc cca ttc tat 768 AlaIle Gln Pro Asn Ile Asn Lys Glu Phe Asp Gly Cys Pro Phe Tyr 245 250 255ggc aat cct tct gat atc gaa tac tgc aaa ggt ctg atg gag cca tac 816 GlyAsn Pro Ser Asp Ile Glu Tyr Cys Lys Gly Leu Met Glu Pro Tyr 260 265 270aga gct gta tgt cgt aac aat atc aac ttc tac tat tac act cta tcc 864 ArgAla Val Cys Arg Asn Asn Ile Asn Phe Tyr Tyr Tyr Thr Leu Ser 275 280 285tgt gcc ttc gct tac tgt atg gga gga gaa gaa aga gct aaa cac gtc 912 CysAla Phe Ala Tyr Cys Met Gly Gly Glu Glu Arg Ala Lys His Val 290 295 300ctt ttc gac tat gtt gag aca tgc gct gcg ccg gaa acg aga gga acg 960 LeuPhe Asp Tyr Val Glu Thr Cys Ala Ala Pro Glu Thr Arg Gly Thr 305 310 315320 tgt gtt tta tca gga cat act ttc tat gac aca ttc gac aaa gca aga 1008Cys Val Leu Ser Gly His Thr Phe Tyr Asp Thr Phe Asp Lys Ala Arg 325 330335 tat caa ttc cag ggc cca tgc aag gag att ctg atg gcc gca gac tgt 1056Tyr Gln Phe Gln Gly Pro Cys Lys Glu Ile Leu Met Ala Ala Asp Cys 340 345350 tac tgg aac aca tgg gat gta aag gtt tca cat aga gac gtc gaa tca 1104Tyr Trp Asn Thr Trp Asp Val Lys Val Ser His Arg Asp Val Glu Ser 355 360365 tac act gag gta gag aaa gta aca atc agg aaa cag tca act gta gta 1152Tyr Thr Glu Val Glu Lys Val Thr Ile Arg Lys Gln Ser Thr Val Val 370 375380 gat ctc att gtg gat ggc aag cag gtc aag gtt gga gga gtg gat gta 1200Asp Leu Ile Val Asp Gly Lys Gln Val Lys Val Gly Gly Val Asp Val 385 390395 400 tct atc ccg tac agc tct gag aac act tcc ata tac tgg cag gat gga1248 Ser Ile Pro Tyr Ser Ser Glu Asn Thr Ser Ile Tyr Trp Gln Asp Gly 405410 415 gac atc ctg acg acg gcc atc cta cct gaa gct ctt gtc gtt aag ttc1296 Asp Ile Leu Thr Thr Ala Ile Leu Pro Glu Ala Leu Val Val Lys Phe 420425 430 aac ttt aag cag ctc ctt gta gtt cat atc aga gat cca ttc gat gca1344 Asn Phe Lys Gln Leu Leu Val Val His Ile Arg Asp Pro Phe Asp Ala 435440 445 aag aca tgc ggc ata tgt ggt aac tat aat caa gat tca act gat gat1392 Lys Thr Cys Gly Ile Cys Gly Asn Tyr Asn Gln Asp Ser Thr Asp Asp 450455 460 ttc ttt gac gca gaa gga gca tgc gct cta acc ccc aac ccc cca gga1440 Phe Phe Asp Ala Glu Gly Ala Cys Ala Leu Thr Pro Asn Pro Pro Gly 465470 475 480 tgt aca gag gaa cag aaa cca gaa gct gag cga ctt tgc aat aatctc 1488 Cys Thr Glu Glu Gln Lys Pro Glu Ala Glu Arg Leu Cys Asn Asn Leu485 490 495 ttt gat tct tct atc gac gag aaa tgt aat gtc tgc tac aag cctgac 1536 Phe Asp Ser Ser Ile Asp Glu Lys Cys Asn Val Cys Tyr Lys Pro Asp500 505 510 cgg att gcc cga tgt atg tac gag tat tgc ctg agg gga caa caagga 1584 Arg Ile Ala Arg Cys Met Tyr Glu Tyr Cys Leu Arg Gly Gln Gln Gly515 520 525 ttt tgt gac cat gct tgg gag ttc aag aaa gaa tgc tac ata aaacat 1632 Phe Cys Asp His Ala Trp Glu Phe Lys Lys Glu Cys Tyr Ile Lys His530 535 540 gga gac act cta gaa gta cca cct gaa tgt caa taaacgtacaaagatacaga 1685 Gly Asp Thr Leu Glu Val Pro Pro Glu Cys Gln 545 550 555agctaaggct actacagcag aagataaaaa agaaactgta gttccttcaa aaaccgtgta 1745ttttatgtac tcattgttta attagagcaa aataaattgt tattatcata acttaaacta 1805aaaaaaaaaa aaaaa 1820 5 958 DNA Aequorea victoria CDS (115)...(702)Apoequorin-encoding gene 5 gggggggggg gggggggggg gggggggggg gggaatgcaattcatctttg catcaaagaa 60 ttacatcaaa tctctagttg atcaactaaa ttgtctcgacaacaacaagc aaac atg 117 Met 1 aca agc aaa caa tac tca gtc aag ctt acatca gac ttc gac aac cca 165 Thr Ser Lys Gln Tyr Ser Val Lys Leu Thr SerAsp Phe Asp Asn Pro 5 10 15 aga tgg att gga cga cac aag cat atg ttc aatttc ctt gat gtc aac 213 Arg Trp Ile Gly Arg His Lys His Met Phe Asn PheLeu Asp Val Asn 20 25 30 cac aat gga aaa atc tct ctt gac gag atg gtc tacaag gca tct gat 261 His Asn Gly Lys Ile Ser Leu Asp Glu Met Val Tyr LysAla Ser Asp 35 40 45 att gtc atc aat aac ctt gga gca aca cct gag caa gccaaa cga cac 309 Ile Val Ile Asn Asn Leu Gly Ala Thr Pro Glu Gln Ala LysArg His 50 55 60 65 aaa gat gct gta gaa gcc ttc ttc gga gga gct gga atgaaa tat ggt 357 Lys Asp Ala Val Glu Ala Phe Phe Gly Gly Ala Gly Met LysTyr Gly 70 75 80 gtg gaa act gat tgg cct gca tat att gaa gga tgg aaa aaattg gct 405 Val Glu Thr Asp Trp Pro Ala Tyr Ile Glu Gly Trp Lys Lys LeuAla 85 90 95 act gat gaa ttg gag aaa tac gcc aaa aac gaa cca acg ctc atccgt 453 Thr Asp Glu Leu Glu Lys Tyr Ala Lys Asn Glu Pro Thr Leu Ile Arg100 105 110 ata tgg ggt gat gct ttg ttt gat atc gtt gac aaa gat caa aatgga 501 Ile Trp Gly Asp Ala Leu Phe Asp Ile Val Asp Lys Asp Gln Asn Gly115 120 125 gcc att aca ctg gat gaa tgg aaa gca tac acc aaa gct gct ggtatc 549 Ala Ile Thr Leu Asp Glu Trp Lys Ala Tyr Thr Lys Ala Ala Gly Ile130 135 140 145 atc caa tca tca gaa gat tgc gag gaa aca ttc aga gtg tgcgat att 597 Ile Gln Ser Ser Glu Asp Cys Glu Glu Thr Phe Arg Val Cys AspIle 150 155 160 gat gaa agt gga caa ctc gat gtt gat gag atg aca aga caacat tta 645 Asp Glu Ser Gly Gln Leu Asp Val Asp Glu Met Thr Arg Gln HisLeu 165 170 175 gga ttt tgg tac acc atg gat cct gct tgc gaa aag ctc tacggt gga 693 Gly Phe Trp Tyr Thr Met Asp Pro Ala Cys Glu Lys Leu Tyr GlyGly 180 185 190 gct gtc ccc taagaagctc tacggtggtg atgcacccta ggaagatgat742 Ala Val Pro 195 gtgattttga ataaaacact gatgaattca atcaaaattttccaaatttt tgaacgattt 802 caatcgtttg tgttgatttt tgtaattagg aacagattaaatcgaatgat tagttgtttt 862 tttaatcaac agaacttaca aatcgaaaaa gtaaaaaaaaaaaaaaaaaa aaaaaaaaaa 922 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaa 958 6591 DNA Aequorea victoria CDS (1)...(588) Recombinant Aequorin AEQ1 6atg acc agc gaa caa tac tca gtc aag ctt aca cca gac ttc gac aac 48 MetThr Ser Glu Gln Tyr Ser Val Lys Leu Thr Pro Asp Phe Asp Asn 1 5 10 15cca aaa tgg att gga cga cac aag cac atg ttt aat ttt ctt gat gtc 96 ProLys Trp Ile Gly Arg His Lys His Met Phe Asn Phe Leu Asp Val 20 25 30 aaccac aat gga agg atc tct ctt gac gag atg gtc tac aag gcg tcc 144 Asn HisAsn Gly Arg Ile Ser Leu Asp Glu Met Val Tyr Lys Ala Ser 35 40 45 gat attgtt ata aac aat ctt gga gca aca cct gaa caa gcc aaa cgt 192 Asp Ile ValIle Asn Asn Leu Gly Ala Thr Pro Glu Gln Ala Lys Arg 50 55 60 cac aaa gatgct gta gaa gcc ttc ttc gga gga gct gga atg aaa tat 240 His Lys Asp AlaVal Glu Ala Phe Phe Gly Gly Ala Gly Met Lys Tyr 65 70 75 80 ggt gta gaaact gaa tgg cct gaa tac atc gaa gga tgg aaa aga ctg 288 Gly Val Glu ThrGlu Trp Pro Glu Tyr Ile Glu Gly Trp Lys Arg Leu 85 90 95 gct tcc gag gaattg aaa agg tat tca aaa aac caa atc aca ctt att 336 Ala Ser Glu Glu LeuLys Arg Tyr Ser Lys Asn Gln Ile Thr Leu Ile 100 105 110 cgt tta tgg ggtgat gca ttg ttc gat atc att gac aaa gac caa aat 384 Arg Leu Trp Gly AspAla Leu Phe Asp Ile Ile Asp Lys Asp Gln Asn 115 120 125 gga gct att tcactg gat gaa tgg aaa gca tac acc aaa tct gat ggc 432 Gly Ala Ile Ser LeuAsp Glu Trp Lys Ala Tyr Thr Lys Ser Asp Gly 130 135 140 atc atc caa tcgtca gaa gat tgc gag gaa aca ttc aga gtg tgc gat 480 Ile Ile Gln Ser SerGlu Asp Cys Glu Glu Thr Phe Arg Val Cys Asp 145 150 155 160 att gat gaaagt gga cag ctc gat gtt gat gag atg aca aga caa cat 528 Ile Asp Glu SerGly Gln Leu Asp Val Asp Glu Met Thr Arg Gln His 165 170 175 tta gga ttttgg tac acc atg gat cct gct tgc gaa aag ctc tac ggt 576 Leu Gly Phe TrpTyr Thr Met Asp Pro Ala Cys Glu Lys Leu Tyr Gly 180 185 190 gga gct gtcccc taa 591 Gly Ala Val Pro 195 7 591 DNA Aequoria victoria CDS(1)...(588) Recombinant Aequorin AEQ2 7 atg acc agc gaa caa tac tca gtcaag ctt aca tca gac ttc gac aac 48 Met Thr Ser Glu Gln Tyr Ser Val LysLeu Thr Ser Asp Phe Asp Asn 1 5 10 15 cca aga tgg att gga cga cac aagcat atg ttc aat ttc ctt gat gtc 96 Pro Arg Trp Ile Gly Arg His Lys HisMet Phe Asn Phe Leu Asp Val 20 25 30 aac cac aat gga aaa atc tct ctt gacgag atg gtc tac aag gca tct 144 Asn His Asn Gly Lys Ile Ser Leu Asp GluMet Val Tyr Lys Ala Ser 35 40 45 gat att gtc atc aat aac ctt gga gca acacct gag caa gcc aaa cga 192 Asp Ile Val Ile Asn Asn Leu Gly Ala Thr ProGlu Gln Ala Lys Arg 50 55 60 cac aaa gat gct gta gaa gcc ttc ttc gga ggagct gga atg aaa tat 240 His Lys Asp Ala Val Glu Ala Phe Phe Gly Gly AlaGly Met Lys Tyr 65 70 75 80 ggt gtg gaa act gat tgg cct gca tat att gaagga tgg aaa aaa ttg 288 Gly Val Glu Thr Asp Trp Pro Ala Tyr Ile Glu GlyTrp Lys Lys Leu 85 90 95 gct act gat gaa ttg gag aaa tac gcc aaa aac gaacca acg ctc atc 336 Ala Thr Asp Glu Leu Glu Lys Tyr Ala Lys Asn Glu ProThr Leu Ile 100 105 110 cgt ata tgg ggt gat gct ttg ttc gat atc gtt gacaaa gat caa aat 384 Arg Ile Trp Gly Asp Ala Leu Phe Asp Ile Val Asp LysAsp Gln Asn 115 120 125 gga gcc att aca ctg gat gaa tgg aaa gca tac accaaa gct gct ggt 432 Gly Ala Ile Thr Leu Asp Glu Trp Lys Ala Tyr Thr LysAla Ala Gly 130 135 140 atc atc caa tca tca gaa gat tgc gag gaa aca ttcaga gtg tgc gat 480 Ile Ile Gln Ser Ser Glu Asp Cys Glu Glu Thr Phe ArgVal Cys Asp 145 150 155 160 att gat gaa agt gga caa ctc gat gtt gat gagatg aca aga caa cat 528 Ile Asp Glu Ser Gly Gln Leu Asp Val Asp Glu MetThr Arg Gln His 165 170 175 tta gga ttt tgg tac acc atg gat cct gct tgcgaa aag ctc tac ggt 576 Leu Gly Phe Trp Tyr Thr Met Asp Pro Ala Cys GluLys Leu Tyr Gly 180 185 190 gga gct gtc ccc taa 591 Gly Ala Val Pro 1958 591 DNA Aequoria victoria CDS (1)...(588) Recombinant Aequorin AEQ3 8atg acc agc gaa caa tac tca gtc aag ctt aca tca gac ttc gac aac 48 MetThr Ser Glu Gln Tyr Ser Val Lys Leu Thr Ser Asp Phe Asp Asn 1 5 10 15cca aga tgg att gga cga cac aag cat atg ttc aat ttc ctt gat gtc 96 ProArg Trp Ile Gly Arg His Lys His Met Phe Asn Phe Leu Asp Val 20 25 30 aaccac aat gga aaa atc tct ctt gac gag atg gtc tac aag gca tct 144 Asn HisAsn Gly Lys Ile Ser Leu Asp Glu Met Val Tyr Lys Ala Ser 35 40 45 gat attgtc atc aat aac ctt gga gca aca cct gag caa gcc aaa cga 192 Asp Ile ValIle Asn Asn Leu Gly Ala Thr Pro Glu Gln Ala Lys Arg 50 55 60 cac aaa gatgct gta gga gac ttc ttc gga gga gct gga atg aaa tat 240 His Lys Asp AlaVal Gly Asp Phe Phe Gly Gly Ala Gly Met Lys Tyr 65 70 75 80 ggt gtg gaaact gat tgg cct gca tac att gaa gga tgg aaa aaa ttg 288 Gly Val Glu ThrAsp Trp Pro Ala Tyr Ile Glu Gly Trp Lys Lys Leu 85 90 95 gct act gat gaattg gag aaa tac gcc aaa aac gaa cca acg ctc atc 336 Ala Thr Asp Glu LeuGlu Lys Tyr Ala Lys Asn Glu Pro Thr Leu Ile 100 105 110 cgt ata tgg ggtgat gct ttg ttc gat atc gtt gac aaa gat caa aat 384 Arg Ile Trp Gly AspAla Leu Phe Asp Ile Val Asp Lys Asp Gln Asn 115 120 125 gga gcc att acactg gat gaa tgg aaa gca tac acc aaa gct gct ggt 432 Gly Ala Ile Thr LeuAsp Glu Trp Lys Ala Tyr Thr Lys Ala Ala Gly 130 135 140 atc atc caa tcatca gaa gat tgc gag gaa aca ttc aga gtg tgc gat 480 Ile Ile Gln Ser SerGlu Asp Cys Glu Glu Thr Phe Arg Val Cys Asp 145 150 155 160 att gat gaaaat gga caa ctc gat gtt gat gag atg aca aga caa cat 528 Ile Asp Glu AsnGly Gln Leu Asp Val Asp Glu Met Thr Arg Gln His 165 170 175 tta gga ttttgg tac acc atg gat cct gct tgc gaa aag ctc tac ggt 576 Leu Gly Phe TrpTyr Thr Met Asp Pro Ala Cys Glu Lys Leu Tyr Gly 180 185 190 gga gct gtcccc taa 591 Gly Ala Val Pro 195 9 567 DNA Aequoria victoria CDS(1)...(567) Aequorin photoprotein 9 gtc aag ctt aca cca gac ttc gac aaccca aaa tgg att gga cga cac 48 Val Lys Leu Thr Pro Asp Phe Asp Asn ProLys Trp Ile Gly Arg His 1 5 10 15 aag cac atg ttt aat ttt ctt gat gtcaac cac aat gga agg atc tct 96 Lys His Met Phe Asn Phe Leu Asp Val AsnHis Asn Gly Arg Ile Ser 20 25 30 ctt gac gag atg gtc tac aag gcg tcc gatatt gtt ata aac aat ctt 144 Leu Asp Glu Met Val Tyr Lys Ala Ser Asp IleVal Ile Asn Asn Leu 35 40 45 gga gca aca cct gaa caa gcc aaa cgt cac aaagat gct gta gaa gcc 192 Gly Ala Thr Pro Glu Gln Ala Lys Arg His Lys AspAla Val Glu Ala 50 55 60 ttc ttc gga gga gct gca atg aaa tat ggt gta gaaact gaa tgg cct 240 Phe Phe Gly Gly Ala Ala Met Lys Tyr Gly Val Glu ThrGlu Trp Pro 65 70 75 80 gaa tac atc gaa gga tgg aaa aga ctg gct tcc gaggaa ttg aaa agg 288 Glu Tyr Ile Glu Gly Trp Lys Arg Leu Ala Ser Glu GluLeu Lys Arg 85 90 95 tat tca aaa aac caa atc aca ctt att cgt tta tgg ggtgat gca ttg 336 Tyr Ser Lys Asn Gln Ile Thr Leu Ile Arg Leu Trp Gly AspAla Leu 100 105 110 ttc gat atc att gac aaa gac caa aat gga gct att tcactg gat gaa 384 Phe Asp Ile Ile Asp Lys Asp Gln Asn Gly Ala Ile Ser LeuAsp Glu 115 120 125 tgg aaa gca tac acc aaa tct gct ggc atc atc caa tcgtca gaa gat 432 Trp Lys Ala Tyr Thr Lys Ser Ala Gly Ile Ile Gln Ser SerGlu Asp 130 135 140 tgc gag gaa aca ttc aga gtg tgc gat att gat gaa agtgga cag ctc 480 Cys Glu Glu Thr Phe Arg Val Cys Asp Ile Asp Glu Ser GlyGln Leu 145 150 155 160 gat gtt gat gag atg aca aga caa cat tta gga ttttgg tac acc atg 528 Asp Val Asp Glu Met Thr Arg Gln His Leu Gly Phe TrpTyr Thr Met 165 170 175 gat cct gct tgc gaa aag ctc tac ggt gga gct gtcccc 567 Asp Pro Ala Cys Glu Lys Leu Tyr Gly Gly Ala Val Pro 180 185 10588 DNA Aequoria victoria CDS (1)...(588) Aequorin mutant w/increasedbioluminescence activity 10 atg acc agc gaa caa tac tca gtc aag ctt acacca gac ttc gac aac 48 Met Thr Ser Glu Gln Tyr Ser Val Lys Leu Thr ProAsp Phe Asp Asn 1 5 10 15 cca aaa tgg att gga cga cac aag cac atg tttaat ttt ctt gat gtc 96 Pro Lys Trp Ile Gly Arg His Lys His Met Phe AsnPhe Leu Asp Val 20 25 30 aac cac aat gga agg atc tct ctt gac gag atg gtctac aag gcg tcc 144 Asn His Asn Gly Arg Ile Ser Leu Asp Glu Met Val TyrLys Ala Ser 35 40 45 gat att gtt ata aac aat ctt gga gca aca cct gaa caagcc aaa cgt 192 Asp Ile Val Ile Asn Asn Leu Gly Ala Thr Pro Glu Gln AlaLys Arg 50 55 60 cac aaa gat gct gta gaa gcc ttc ttc gga gga gct gca atgaaa tat 240 His Lys Asp Ala Val Glu Ala Phe Phe Gly Gly Ala Ala Met LysTyr 65 70 75 80 ggt gta gaa act gaa tgg cct gaa tac atc gaa gga tgg aaaaga ctg 288 Gly Val Glu Thr Glu Trp Pro Glu Tyr Ile Glu Gly Trp Lys ArgLeu 85 90 95 gct tcc gag gaa ttg aaa agg tat tca aaa aac caa atc aca cttatt 336 Ala Ser Glu Glu Leu Lys Arg Tyr Ser Lys Asn Gln Ile Thr Leu Ile100 105 110 cgt tta tgg ggt gat gca ttg ttc gat atc att tcc aaa gac caaaat 384 Arg Leu Trp Gly Asp Ala Leu Phe Asp Ile Ile Ser Lys Asp Gln Asn115 120 125 gga gct att tca ctg gat gaa tgg aaa gca tac acc aaa tct gctggc 432 Gly Ala Ile Ser Leu Asp Glu Trp Lys Ala Tyr Thr Lys Ser Ala Gly130 135 140 atc atc caa tcg tca gaa gat tgc gag gaa aca ttc aga gtg tgcgat 480 Ile Ile Gln Ser Ser Glu Asp Cys Glu Glu Thr Phe Arg Val Cys Asp145 150 155 160 att gat gaa agt gga cag ctc gat gtt gat gag atg aca agacaa cat 528 Ile Asp Glu Ser Gly Gln Leu Asp Val Asp Glu Met Thr Arg GlnHis 165 170 175 tta gga ttt tgg tac acc atg gat cct gct tgc gaa aag ctctac ggt 576 Leu Gly Phe Trp Tyr Thr Met Asp Pro Ala Cys Glu Lys Leu TyrGly 180 185 190 gga gct gtc ccc 588 Gly Ala Val Pro 195 11 588 DNAAequorea victoria CDS (1)...(588) Recombinant site-directed Aequorinmutant 11 atg acc agc gaa caa tac tca gtc aag ctt aca cca gac ttc gacaac 48 Met Thr Ser Glu Gln Tyr Ser Val Lys Leu Thr Pro Asp Phe Asp Asn 15 10 15 cca aaa tgg att gga cga cac aag cac atg ttt aat ttt ctt gat gtc96 Pro Lys Trp Ile Gly Arg His Lys His Met Phe Asn Phe Leu Asp Val 20 2530 aac cac aat gga agg atc tct ctt gac gag atg gtc tac aag gcg tcc 144Asn His Asn Gly Arg Ile Ser Leu Asp Glu Met Val Tyr Lys Ala Ser 35 40 45gat att gtt ata aac aat ctt gga gca aca cct gaa caa gcc aaa cgt 192 AspIle Val Ile Asn Asn Leu Gly Ala Thr Pro Glu Gln Ala Lys Arg 50 55 60 cacaaa gat gct gta gaa gcc ttc ttc gga gga gct gca atg aaa tat 240 His LysAsp Ala Val Glu Ala Phe Phe Gly Gly Ala Ala Met Lys Tyr 65 70 75 80 ggtgta gaa act gaa tgg cct gaa tac atc gaa gga tgg aaa aga ctg 288 Gly ValGlu Thr Glu Trp Pro Glu Tyr Ile Glu Gly Trp Lys Arg Leu 85 90 95 gct tccgag gaa ttg aaa agg tat tca aaa aac caa atc aca ctt att 336 Ala Ser GluGlu Leu Lys Arg Tyr Ser Lys Asn Gln Ile Thr Leu Ile 100 105 110 cgt ttatgg ggt gat gca ttg ttc gat atc att tcc aaa gac caa aat 384 Arg Leu TrpGly Asp Ala Leu Phe Asp Ile Ile Ser Lys Asp Gln Asn 115 120 125 gga gctatt tca ctg gat tca tgg aaa gca tac acc aaa tct gct ggc 432 Gly Ala IleSer Leu Asp Ser Trp Lys Ala Tyr Thr Lys Ser Ala Gly 130 135 140 atc atccaa tcg tca gaa gat tgc gag gaa aca ttc aga gtg tgc gat 480 Ile Ile GlnSer Ser Glu Asp Cys Glu Glu Thr Phe Arg Val Cys Asp 145 150 155 160 attgat gaa agt gga cag ctc gat gtt gat gag atg aca aga caa cat 528 Ile AspGlu Ser Gly Gln Leu Asp Val Asp Glu Met Thr Arg Gln His 165 170 175 ttagga ttt tgg tac acc atg gat cct gct tgc gaa aag ctc tac ggt 576 Leu GlyPhe Trp Tyr Thr Met Asp Pro Ala Cys Glu Lys Leu Tyr Gly 180 185 190 ggagct gtc ccc 588 Gly Ala Val Pro 195 12 588 DNA Aequorea victoria CDS(1)...(588) Aequorin mutant with increased biolumenescence activity 12atg acc agc gaa caa tac tca gtc aag ctt aca cca gac ttc gac aac 48 MetThr Ser Glu Gln Tyr Ser Val Lys Leu Thr Pro Asp Phe Asp Asn 1 5 10 15cca aaa tgg att gga cga cac aag cac atg ttt aat ttt ctt gat gtc 96 ProLys Trp Ile Gly Arg His Lys His Met Phe Asn Phe Leu Asp Val 20 25 30 aaccac aat gga agg atc tct ctt gac gag atg gtc tac aag gcg tcc 144 Asn HisAsn Gly Arg Ile Ser Leu Asp Glu Met Val Tyr Lys Ala Ser 35 40 45 gat attgtt ata aac aat ctt gga gca aca cct gaa caa gcc aaa cgt 192 Asp Ile ValIle Asn Asn Leu Gly Ala Thr Pro Glu Gln Ala Lys Arg 50 55 60 cac aaa gatgct gta gaa gcc ttc ttc gga gga gct gca atg aaa tat 240 His Lys Asp AlaVal Glu Ala Phe Phe Gly Gly Ala Ala Met Lys Tyr 65 70 75 80 ggt gta gaaact gaa tgg cct gaa tac atc gaa gga tgg aaa aga ctg 288 Gly Val Glu ThrGlu Trp Pro Glu Tyr Ile Glu Gly Trp Lys Arg Leu 85 90 95 gct tcc gag gaattg aaa agg tat tca aaa aac caa atc aca ctt att 336 Ala Ser Glu Glu LeuLys Arg Tyr Ser Lys Asn Gln Ile Thr Leu Ile 100 105 110 cgt tta tgg ggtgat gca ttg ttc gat atc att tcc aaa gac caa aat 384 Arg Leu Trp Gly AspAla Leu Phe Asp Ile Ile Ser Lys Asp Gln Asn 115 120 125 gca gct att tcactg gat gaa tgg aaa gca tac acc aaa tct gct ggc 432 Ala Ala Ile Ser LeuAsp Glu Trp Lys Ala Tyr Thr Lys Ser Ala Gly 130 135 140 atc atc caa tcgtca gaa gat tgc gag gaa aca ttc aga gtg tgc gat 480 Ile Ile Gln Ser SerGlu Asp Cys Glu Glu Thr Phe Arg Val Cys Asp 145 150 155 160 att gat gaaagt gga cag ctc gat gtt gat gag atg aca aga caa cat 528 Ile Asp Glu SerGly Gln Leu Asp Val Asp Glu Met Thr Arg Gln His 165 170 175 tta gga ttttgg tac acc atg gat cct gct tgc gaa aag ctc tac ggt 576 Leu Gly Phe TrpTyr Thr Met Asp Pro Ala Cys Glu Lys Leu Tyr Gly 180 185 190 gga gct gtcccc 588 Gly Ala Val Pro 195 13 567 DNA Aequorea victoria CDS (1)...(567)Recombinant apoaequorin (AQUALITEp) 13 gtc aag ctt aca cca gac ttc gacaac cca aaa tgg att gga cga cac 48 Val Lys Leu Thr Pro Asp Phe Asp AsnPro Lys Trp Ile Gly Arg His 1 5 10 15 aag cac atg ttt aat ttt ctt gatgtc aac cac aat gga agg atc tct 96 Lys His Met Phe Asn Phe Leu Asp ValAsn His Asn Gly Arg Ile Ser 20 25 30 ctt gac gag atg gtc tac aag gcg tccgat att gtt ata aac aat ctt 144 Leu Asp Glu Met Val Tyr Lys Ala Ser AspIle Val Ile Asn Asn Leu 35 40 45 gga gca aca cct gaa caa gcc aaa cgt cacaaa gat gct gta gaa gcc 192 Gly Ala Thr Pro Glu Gln Ala Lys Arg His LysAsp Ala Val Glu Ala 50 55 60 ttc ttc gga gga gct gga atg aaa tat ggt gtagaa act gaa tgg cct 240 Phe Phe Gly Gly Ala Gly Met Lys Tyr Gly Val GluThr Glu Trp Pro 65 70 75 80 gaa tac atc gaa gga tgg aaa aaa ctg gct tccgag gaa ttg aaa agg 288 Glu Tyr Ile Glu Gly Trp Lys Lys Leu Ala Ser GluGlu Leu Lys Arg 85 90 95 tat tca aaa aac caa atc aca ctt att cgt tta tggggt gat gca ttg 336 Tyr Ser Lys Asn Gln Ile Thr Leu Ile Arg Leu Trp GlyAsp Ala Leu 100 105 110 ttc gat atc att gac aaa gac caa aat gga gct attctg tca gat gaa 384 Phe Asp Ile Ile Asp Lys Asp Gln Asn Gly Ala Ile LeuSer Asp Glu 115 120 125 tgg aaa gca tac acc aaa tct gat ggc atc atc caatcg tca gaa gat 432 Trp Lys Ala Tyr Thr Lys Ser Asp Gly Ile Ile Gln SerSer Glu Asp 130 135 140 tgc gag gaa aca ttc aga gtg tgc gat att gat gaaagt gga cag ctc 480 Cys Glu Glu Thr Phe Arg Val Cys Asp Ile Asp Glu SerGly Gln Leu 145 150 155 160 gat gtt gat gag atg aca aga caa cat tta ggattt tgg tac acc atg 528 Asp Val Asp Glu Met Thr Arg Gln His Leu Gly PheTrp Tyr Thr Met 165 170 175 gat cct gct tgc gaa aag ctc tac ggt gga gctgtc ccc 567 Asp Pro Ala Cys Glu Lys Leu Tyr Gly Gly Ala Val Pro 180 18514 236 PRT Vibrio fisheri 14 Met Pro Ile Asn Cys Lys Val Lys Ser Ile GluPro Leu Ala Cys Asn 1 5 10 15 Thr Phe Arg Ile Leu Leu His Pro Glu GlnPro Val Ala Phe Lys Ala 20 25 30 Gly Gln Tyr Leu Thr Val Val Met Gly GluLys Asp Lys Arg Pro Phe 35 40 45 Ser Ile Ala Ser Ser Pro Cys Arg His GluGly Glu Ile Glu Leu His 50 55 60 Ile Gly Ala Ala Glu His Asn Ala Tyr AlaGly Glu Val Val Glu Ser 65 70 75 80 Met Lys Ser Ala Leu Glu Thr Gly GlyAsp Ile Leu Ile Asp Ala Pro 85 90 95 His Gly Glu Ala Trp Ile Arg Glu AspSer Asp Arg Ser Met Leu Leu 100 105 110 Ile Ala Gly Gly Thr Gly Phe SerTyr Val Arg Ser Ile Leu Asp His 115 120 125 Cys Ile Ser Gln Gln Ile GlnLys Pro Ile Tyr Leu Tyr Trp Gly Gly 130 135 140 Arg Asp Glu Cys Gln LeuTyr Ala Lys Ala Glu Leu Glu Ser Ile Ala 145 150 155 160 Gln Ala His SerHis Ile Thr Phe Val Pro Val Val Glu Lys Ser Glu 165 170 175 Gly Trp ThrGly Lys Thr Gly Asn Val Leu Glu Ala Val Lys Ala Asp 180 185 190 Phe AsnSer Leu Ala Asp Met Asp Ile Tyr Ile Ala Gly Arg Phe Glu 195 200 205 MetAla Gly Ala Ala Arg Glu Gln Phe Thr Thr Glu Lys Gln Ala Lys 210 215 220Lys Glu Gln Leu Phe Gly Asp Ala Phe Ala Phe Ile 225 230 235 15 1079 DNARenilla mulleri CDS (259)...(975) Renilla mulleri GFP 15 ggttatacacaagtgtatcg cgtatctgca gacgcatcta gtgggattat tcgagcggta 60 gtatttacgtcagacctgtc taatcgaaac cacaacaaac tcttaaaata agccacattt 120 acataatatctaagagacgc ctcatttaag agtagtaaaa atataatata tgatagagta 180 tacaactctcgccttagaca gacagtgtgc aacagagtaa ctcttgttaa tgcaatcgaa 240 agcgtcaagagagataag atg agt aaa caa ata ttg aag aac act tgt tta 291 Met Ser Lys GlnIle Leu Lys Asn Thr Cys Leu 1 5 10 caa gaa gta atg tcg tat aaa gta aatctg gaa gga att gta aac aac 339 Gln Glu Val Met Ser Tyr Lys Val Asn LeuGlu Gly Ile Val Asn Asn 15 20 25 cat gtt ttt aca atg gag ggt tgc ggc aaaggg aat att tta ttc ggc 387 His Val Phe Thr Met Glu Gly Cys Gly Lys GlyAsn Ile Leu Phe Gly 30 35 40 aat caa ctg gtt cag att cgt gtc acg aaa ggggcc cca ctg cct ttt 435 Asn Gln Leu Val Gln Ile Arg Val Thr Lys Gly AlaPro Leu Pro Phe 45 50 55 gca ttt gat att gtg tca cca gct ttt caa tat ggcaac cgt act ttc 483 Ala Phe Asp Ile Val Ser Pro Ala Phe Gln Tyr Gly AsnArg Thr Phe 60 65 70 75 acg aaa tat ccg aat gat ata tca gat tat ttt atacaa tca ttt cca 531 Thr Lys Tyr Pro Asn Asp Ile Ser Asp Tyr Phe Ile GlnSer Phe Pro 80 85 90 gca gga ttt atg tat gaa cga aca tta cgt tac gaa gatggc gga ctt 579 Ala Gly Phe Met Tyr Glu Arg Thr Leu Arg Tyr Glu Asp GlyGly Leu 95 100 105 gtt gaa att cgt tca gat ata aat tta ata gaa gac aagttc gtc tac 627 Val Glu Ile Arg Ser Asp Ile Asn Leu Ile Glu Asp Lys PheVal Tyr 110 115 120 aga gtg gaa tac aaa ggt agt aac ttc cca gat gat ggtccc gtc atg 675 Arg Val Glu Tyr Lys Gly Ser Asn Phe Pro Asp Asp Gly ProVal Met 125 130 135 cag aag act atc tta gga ata gag cct tca ttt gaa gccatg tac atg 723 Gln Lys Thr Ile Leu Gly Ile Glu Pro Ser Phe Glu Ala MetTyr Met 140 145 150 155 aat aat ggc gtc ttg gtc ggc gaa gta att ctt gtctat aaa cta aac 771 Asn Asn Gly Val Leu Val Gly Glu Val Ile Leu Val TyrLys Leu Asn 160 165 170 tct ggg aaa tat tat tca tgt cac atg aaa aca ttaatg aag tcg aaa 819 Ser Gly Lys Tyr Tyr Ser Cys His Met Lys Thr Leu MetLys Ser Lys 175 180 185 ggt gta gta aag gag ttt cct tcg tat cat ttt attcaa cat cgt ttg 867 Gly Val Val Lys Glu Phe Pro Ser Tyr His Phe Ile GlnHis Arg Leu 190 195 200 gaa aag act tac gta gaa gac ggg ggg ttc gtt gaacag cat gag act 915 Glu Lys Thr Tyr Val Glu Asp Gly Gly Phe Val Glu GlnHis Glu Thr 205 210 215 gct att gct caa atg aca tct ata gga aaa cca ctagga tcc tta cac 963 Ala Ile Ala Gln Met Thr Ser Ile Gly Lys Pro Leu GlySer Leu His 220 225 230 235 gaa tgg gtt taa acacagttac attactttttccaattcgtg tttcatgtca 1015 Glu Trp Val * aataataatt ttttaaacaattatcaatgt tttgtgatat gtttgtaaaa aaaaaaaaaa 1075 aaaa 1079 16 238 PRTRenilla mulleri 16 Met Ser Lys Gln Ile Leu Lys Asn Thr Cys Leu Gln GluVal Met Ser 1 5 10 15 Tyr Lys Val Asn Leu Glu Gly Ile Val Asn Asn HisVal Phe Thr Met 20 25 30 Glu Gly Cys Gly Lys Gly Asn Ile Leu Phe Gly AsnGln Leu Val Gln 35 40 45 Ile Arg Val Thr Lys Gly Ala Pro Leu Pro Phe AlaPhe Asp Ile Val 50 55 60 Ser Pro Ala Phe Gln Tyr Gly Asn Arg Thr Phe ThrLys Tyr Pro Asn 65 70 75 80 Asp Ile Ser Asp Tyr Phe Ile Gln Ser Phe ProAla Gly Phe Met Tyr 85 90 95 Glu Arg Thr Leu Arg Tyr Glu Asp Gly Gly LeuVal Glu Ile Arg Ser 100 105 110 Asp Ile Asn Leu Ile Glu Asp Lys Phe ValTyr Arg Val Glu Tyr Lys 115 120 125 Gly Ser Asn Phe Pro Asp Asp Gly ProVal Met Gln Lys Thr Ile Leu 130 135 140 Gly Ile Glu Pro Ser Phe Glu AlaMet Tyr Met Asn Asn Gly Val Leu 145 150 155 160 Val Gly Glu Val Ile LeuVal Tyr Lys Leu Asn Ser Gly Lys Tyr Tyr 165 170 175 Ser Cys His Met LysThr Leu Met Lys Ser Lys Gly Val Val Lys Glu 180 185 190 Phe Pro Ser TyrHis Phe Ile Gln His Arg Leu Glu Lys Thr Tyr Val 195 200 205 Glu Asp GlyGly Phe Val Glu Gln His Glu Thr Ala Ile Ala Gln Met 210 215 220 Thr SerIle Gly Lys Pro Leu Gly Ser Leu His Glu Trp Val 225 230 235 17 1217 DNARenilla mulleri CDS (31)...(963) Renilla mulleri luciferase 17cggcacgagg tttaagaatc aataaaaaaa atg acg tca aaa gtt tac gat cct 54 MetThr Ser Lys Val Tyr Asp Pro 1 5 gaa tta aga aaa cgc atg att act ggt ccacaa tgg tgg gca aga tgt 102 Glu Leu Arg Lys Arg Met Ile Thr Gly Pro GlnTrp Trp Ala Arg Cys 10 15 20 aaa caa atg aat gtt ctt gat tca ttt att aattat tat gat tca gaa 150 Lys Gln Met Asn Val Leu Asp Ser Phe Ile Asn TyrTyr Asp Ser Glu 25 30 35 40 aaa cat gca gaa aat gca gtt ata ttt tta catggt aat gca gca tct 198 Lys His Ala Glu Asn Ala Val Ile Phe Leu His GlyAsn Ala Ala Ser 45 50 55 tct tat tta tgg cgt cat gtt gta cca cat gtt gaacca gtg gcg cga 246 Ser Tyr Leu Trp Arg His Val Val Pro His Val Glu ProVal Ala Arg 60 65 70 tgt att ata cca gat ctt ata ggt atg ggt aaa tca ggcaag tct ggt 294 Cys Ile Ile Pro Asp Leu Ile Gly Met Gly Lys Ser Gly LysSer Gly 75 80 85 aat ggt tcc tat aga tta cta gat cat tac aaa tat ctt actgaa tgg 342 Asn Gly Ser Tyr Arg Leu Leu Asp His Tyr Lys Tyr Leu Thr GluTrp 90 95 100 ttc aaa cat ctt aat tta cca aag aag atc att ttt gtc ggtcat gat 390 Phe Lys His Leu Asn Leu Pro Lys Lys Ile Ile Phe Val Gly HisAsp 105 110 115 120 tgg ggt gct tgt tta gca ttt cat tat tgc tat gaa catcag gat cgc 438 Trp Gly Ala Cys Leu Ala Phe His Tyr Cys Tyr Glu His GlnAsp Arg 125 130 135 atc aaa gca gtt gtt cat gct gaa agt gta gta gat gtgatt gaa tcg 486 Ile Lys Ala Val Val His Ala Glu Ser Val Val Asp Val IleGlu Ser 140 145 150 tgg gac gaa tgg cct gat att gaa gaa gat att gct ttgatt aaa tct 534 Trp Asp Glu Trp Pro Asp Ile Glu Glu Asp Ile Ala Leu IleLys Ser 155 160 165 gaa gaa gga gaa aaa atg gtt tta gag aat aac ttc ttcgtg gaa acc 582 Glu Glu Gly Glu Lys Met Val Leu Glu Asn Asn Phe Phe ValGlu Thr 170 175 180 atg ttg cca tca aaa atc atg aga aag ttg gaa cca gaggaa ttt gct 630 Met Leu Pro Ser Lys Ile Met Arg Lys Leu Glu Pro Glu GluPhe Ala 185 190 195 200 gct tat ctt gaa cca ttt aaa gag aaa ggt gaa gttcgt cgt cca aca 678 Ala Tyr Leu Glu Pro Phe Lys Glu Lys Gly Glu Val ArgArg Pro Thr 205 210 215 tta tca tgg cct cgt gaa atc cct ttg gta aaa ggtggt aaa ccg gat 726 Leu Ser Trp Pro Arg Glu Ile Pro Leu Val Lys Gly GlyLys Pro Asp 220 225 230 gta gta gaa att gtc agg aat tat aat gct tat cttcgt gca agt cat 774 Val Val Glu Ile Val Arg Asn Tyr Asn Ala Tyr Leu ArgAla Ser His 235 240 245 gat tta cca aaa atg ttt att gaa tct gat cca ggattc ttt tcc aat 822 Asp Leu Pro Lys Met Phe Ile Glu Ser Asp Pro Gly PhePhe Ser Asn 250 255 260 gct att gtt gaa ggt gct aag aaa ttc cct aat actgaa ttt gtt aaa 870 Ala Ile Val Glu Gly Ala Lys Lys Phe Pro Asn Thr GluPhe Val Lys 265 270 275 280 gtc aaa ggt ctt cat ttt tca caa gaa gat gcacct gat gaa atg gga 918 Val Lys Gly Leu His Phe Ser Gln Glu Asp Ala ProAsp Glu Met Gly 285 290 295 aat tat ata aaa tcg ttt gtt gag cgt gtt cttaaa aat gaa caa 963 Asn Tyr Ile Lys Ser Phe Val Glu Arg Val Leu Lys AsnGlu Gln 300 305 310 taaactacca ggtttccatg ttgccacttt agctgggtttaataaatttc actatcaatt 1023 tgaacaattt cacattaatt ttaactatta aaaaattatggacacaggga ttatatcaga 1083 tgattaattt agttgggaac aatgaatacc gaatattatgaattctcttt agctatttat 1143 aataatcaca ttcttatgta ataaaacttt gttttaataaattaatgatt cagaaaaaaa 1203 aaaaaaaaaa aaaa 1217 18 311 PRT Renillamulleri 18 Met Thr Ser Lys Val Tyr Asp Pro Glu Leu Arg Lys Arg Met IleThr 1 5 10 15 Gly Pro Gln Trp Trp Ala Arg Cys Lys Gln Met Asn Val LeuAsp Ser 20 25 30 Phe Ile Asn Tyr Tyr Asp Ser Glu Lys His Ala Glu Asn AlaVal Ile 35 40 45 Phe Leu His Gly Asn Ala Ala Ser Ser Tyr Leu Trp Arg HisVal Val 50 55 60 Pro His Val Glu Pro Val Ala Arg Cys Ile Ile Pro Asp LeuIle Gly 65 70 75 80 Met Gly Lys Ser Gly Lys Ser Gly Asn Gly Ser Tyr ArgLeu Leu Asp 85 90 95 His Tyr Lys Tyr Leu Thr Glu Trp Phe Lys His Leu AsnLeu Pro Lys 100 105 110 Lys Ile Ile Phe Val Gly His Asp Trp Gly Ala CysLeu Ala Phe His 115 120 125 Tyr Cys Tyr Glu His Gln Asp Arg Ile Lys AlaVal Val His Ala Glu 130 135 140 Ser Val Val Asp Val Ile Glu Ser Trp AspGlu Trp Pro Asp Ile Glu 145 150 155 160 Glu Asp Ile Ala Leu Ile Lys SerGlu Glu Gly Glu Lys Met Val Leu 165 170 175 Glu Asn Asn Phe Phe Val GluThr Met Leu Pro Ser Lys Ile Met Arg 180 185 190 Lys Leu Glu Pro Glu GluPhe Ala Ala Tyr Leu Glu Pro Phe Lys Glu 195 200 205 Lys Gly Glu Val ArgArg Pro Thr Leu Ser Trp Pro Arg Glu Ile Pro 210 215 220 Leu Val Lys GlyGly Lys Pro Asp Val Val Glu Ile Val Arg Asn Tyr 225 230 235 240 Asn AlaTyr Leu Arg Ala Ser His Asp Leu Pro Lys Met Phe Ile Glu 245 250 255 SerAsp Pro Gly Phe Phe Ser Asn Ala Ile Val Glu Gly Ala Lys Lys 260 265 270Phe Pro Asn Thr Glu Phe Val Lys Val Lys Gly Leu His Phe Ser Gln 275 280285 Glu Asp Ala Pro Asp Glu Met Gly Asn Tyr Ile Lys Ser Phe Val Glu 290295 300 Arg Val Leu Lys Asn Glu Gln 305 310 19 765 DNA Gaussia CDS(37)...(594) 19 gcacgagggt actcaaagta tcttctggca gggaaa atg gga gtg aaagtt ctt 54 Met Gly Val Lys Val Leu 1 5 ttt gcc ctt att tgt att gct gtggcc gag gcc aaa cca act gaa aac 102 Phe Ala Leu Ile Cys Ile Ala Val AlaGlu Ala Lys Pro Thr Glu Asn 10 15 20 aat gaa gat ttc aac att gta gct gtagct agc aac ttt gct aca acg 150 Asn Glu Asp Phe Asn Ile Val Ala Val AlaSer Asn Phe Ala Thr Thr 25 30 35 gat ctc gat gct gac cgt ggt aaa ttg cccgga aaa aaa tta cca ctt 198 Asp Leu Asp Ala Asp Arg Gly Lys Leu Pro GlyLys Lys Leu Pro Leu 40 45 50 gag gta ctc aaa gaa atg gaa gcc aat gct aggaaa gct ggc tgc act 246 Glu Val Leu Lys Glu Met Glu Ala Asn Ala Arg LysAla Gly Cys Thr 55 60 65 70 agg gga tgt ctg ata tgc ctg tca cac atc aagtgt aca ccc aaa atg 294 Arg Gly Cys Leu Ile Cys Leu Ser His Ile Lys CysThr Pro Lys Met 75 80 85 aag aag ttt atc cca gga aga tgc cac acc tat gaagga gac aaa gaa 342 Lys Lys Phe Ile Pro Gly Arg Cys His Thr Tyr Glu GlyAsp Lys Glu 90 95 100 agt gca cag gga gga ata gga gag gct att gtt gacatt cct gaa att 390 Ser Ala Gln Gly Gly Ile Gly Glu Ala Ile Val Asp IlePro Glu Ile 105 110 115 cct ggg ttt aag gat ttg gaa ccc atg gaa caa ttcatt gca caa gtt 438 Pro Gly Phe Lys Asp Leu Glu Pro Met Glu Gln Phe IleAla Gln Val 120 125 130 gac cta tgt gta gac tgc aca act gga tgc ctc aaaggt ctt gcc aat 486 Asp Leu Cys Val Asp Cys Thr Thr Gly Cys Leu Lys GlyLeu Ala Asn 135 140 145 150 gtg caa tgt tct gat tta ctc aag aaa tgg ctgcca caa aga tgt gca 534 Val Gln Cys Ser Asp Leu Leu Lys Lys Trp Leu ProGln Arg Cys Ala 155 160 165 act ttt gct agc aaa att caa ggc caa gtg gacaaa ata aag ggt gcc 582 Thr Phe Ala Ser Lys Ile Gln Gly Gln Val Asp LysIle Lys Gly Ala 170 175 180 ggt ggt gat taa tcctaataga atactgcataactggatgat gatatactag 634 Gly Gly Asp * 185 cttattgctc ataaaatggccattttttgt aacaaatcga gtctatgtaa ttcaaaatac 694 ctaattaatt gttaatacatatgtaattcc tataaatata atttatgcaa tccaaaaaaa 754 aaaaaaaaaa a 765 20 185PRT Renilla mulleri 20 Met Gly Val Lys Val Leu Phe Ala Leu Ile Cys IleAla Val Ala Glu 1 5 10 15 Ala Lys Pro Thr Glu Asn Asn Glu Asp Phe AsnIle Val Ala Val Ala 20 25 30 Ser Asn Phe Ala Thr Thr Asp Leu Asp Ala AspArg Gly Lys Leu Pro 35 40 45 Gly Lys Lys Leu Pro Leu Glu Val Leu Lys GluMet Glu Ala Asn Ala 50 55 60 Arg Lys Ala Gly Cys Thr Arg Gly Cys Leu IleCys Leu Ser His Ile 65 70 75 80 Lys Cys Thr Pro Lys Met Lys Lys Phe IlePro Gly Arg Cys His Thr 85 90 95 Tyr Glu Gly Asp Lys Glu Ser Ala Gln GlyGly Ile Gly Glu Ala Ile 100 105 110 Val Asp Ile Pro Glu Ile Pro Gly PheLys Asp Leu Glu Pro Met Glu 115 120 125 Gln Phe Ile Ala Gln Val Asp LeuCys Val Asp Cys Thr Thr Gly Cys 130 135 140 Leu Lys Gly Leu Ala Asn ValGln Cys Ser Asp Leu Leu Lys Lys Trp 145 150 155 160 Leu Pro Gln Arg CysAla Thr Phe Ala Ser Lys Ile Gln Gly Gln Val 165 170 175 Asp Lys Ile LysGly Ala Gly Gly Asp 180 185 21 1146 DNA Gaussia CDS (1)...(1146)Nucleotide sequence encoding a CBD-Gaussia luciferase fusion protein 21atg tca gtt gaa ttt tac aac tct aac aaa tca gca caa aca aac tca 48 MetSer Val Glu Phe Tyr Asn Ser Asn Lys Ser Ala Gln Thr Asn Ser 1 5 10 15att aca cca ata atc aaa att act aac aca tct gac agt gat tta aat 96 IleThr Pro Ile Ile Lys Ile Thr Asn Thr Ser Asp Ser Asp Leu Asn 20 25 30 ttaaat gac gta aaa gtt aga tat tat tac aca agt gat ggt aca caa 144 Leu AsnAsp Val Lys Val Arg Tyr Tyr Tyr Thr Ser Asp Gly Thr Gln 35 40 45 gga caaact ttc tgg tgt gac cat gct ggt gca tta tta gga aat agc 192 Gly Gln ThrPhe Trp Cys Asp His Ala Gly Ala Leu Leu Gly Asn Ser 50 55 60 tat gtt gataac act agc aaa gtg aca gca aac ttc gtt aaa gaa aca 240 Tyr Val Asp AsnThr Ser Lys Val Thr Ala Asn Phe Val Lys Glu Thr 65 70 75 80 gca agc ccaaca tca acc tat gat aca tat gtt gaa ttt gga ttt gca 288 Ala Ser Pro ThrSer Thr Tyr Asp Thr Tyr Val Glu Phe Gly Phe Ala 85 90 95 agc gga gca gctact ctt aaa aaa gga caa ttt ata act att caa gga 336 Ser Gly Ala Ala ThrLeu Lys Lys Gly Gln Phe Ile Thr Ile Gln Gly 100 105 110 aga ata aca aaatca gac tgg tca aac tac act caa aca aat gac tat 384 Arg Ile Thr Lys SerAsp Trp Ser Asn Tyr Thr Gln Thr Asn Asp Tyr 115 120 125 tca ttt gat gcaagt agt tca aca cca gtt gta aat cca aaa gtt aca 432 Ser Phe Asp Ala SerSer Ser Thr Pro Val Val Asn Pro Lys Val Thr 130 135 140 gga tat ata ggtgga gct aaa gtt ctt ggt aca gca cca ggt tcc gcg 480 Gly Tyr Ile Gly GlyAla Lys Val Leu Gly Thr Ala Pro Gly Ser Ala 145 150 155 160 ggt ctg gtgcca cgc ggt agt act gca att ggt atg aaa gaa acc gct 528 Gly Leu Val ProArg Gly Ser Thr Ala Ile Gly Met Lys Glu Thr Ala 165 170 175 gct gct aaattc gaa cgc cag cac atg gac agc cca gat ctg ggt acc 576 Ala Ala Lys PheGlu Arg Gln His Met Asp Ser Pro Asp Leu Gly Thr 180 185 190 gat gac gacgac aag atg gga gtg aaa gtt ctt ttt gcc ctt att tgt 624 Asp Asp Asp AspLys Met Gly Val Lys Val Leu Phe Ala Leu Ile Cys 195 200 205 att gct gtggcc gag gcc aaa cca act gaa aac aat gaa gat ttc aac 672 Ile Ala Val AlaGlu Ala Lys Pro Thr Glu Asn Asn Glu Asp Phe Asn 210 215 220 att gta gctgta gct agc aac ttt gct aca acg gat ctc gat gct gac 720 Ile Val Ala ValAla Ser Asn Phe Ala Thr Thr Asp Leu Asp Ala Asp 225 230 235 240 cgt ggtaaa ttg ccc gga aaa aaa tta cca ctt gag gta ctc aaa gaa 768 Arg Gly LysLeu Pro Gly Lys Lys Leu Pro Leu Glu Val Leu Lys Glu 245 250 255 atg gaagcc aat gct agg aaa gct ggc tgc act agg gga tgt ctg ata 816 Met Glu AlaAsn Ala Arg Lys Ala Gly Cys Thr Arg Gly Cys Leu Ile 260 265 270 tgc ctgtca cac atc aag tgt aca ccc aaa atg aag aag ttt atc cca 864 Cys Leu SerHis Ile Lys Cys Thr Pro Lys Met Lys Lys Phe Ile Pro 275 280 285 gga agatgc cac acc tat gaa gga gac aaa gaa agt gca cag gga gga 912 Gly Arg CysHis Thr Tyr Glu Gly Asp Lys Glu Ser Ala Gln Gly Gly 290 295 300 ata ggagag gct att gtt gac att cct gaa att cct ggg ttt aag gat 960 Ile Gly GluAla Ile Val Asp Ile Pro Glu Ile Pro Gly Phe Lys Asp 305 310 315 320 ttggaa ccc atg gaa caa ttc att gca caa gtt gac cta tgt gta gac 1008 Leu GluPro Met Glu Gln Phe Ile Ala Gln Val Asp Leu Cys Val Asp 325 330 335 tgcaca act gga tgc ctc aaa ggt ctt gcc aat gtg caa tgt tct gat 1056 Cys ThrThr Gly Cys Leu Lys Gly Leu Ala Asn Val Gln Cys Ser Asp 340 345 350 ttactc aag aaa tgg ctg cca caa aga tgt gca act ttt gct agc aaa 1104 Leu LeuLys Lys Trp Leu Pro Gln Arg Cys Ala Thr Phe Ala Ser Lys 355 360 365 attcaa ggc caa gtg gac aaa ata aag ggt gcc ggt ggt gat 1146 Ile Gln Gly GlnVal Asp Lys Ile Lys Gly Ala Gly Gly Asp 370 375 380 22 382 PRT Gaussia22 Met Ser Val Glu Phe Tyr Asn Ser Asn Lys Ser Ala Gln Thr Asn Ser 1 510 15 Ile Thr Pro Ile Ile Lys Ile Thr Asn Thr Ser Asp Ser Asp Leu Asn 2025 30 Leu Asn Asp Val Lys Val Arg Tyr Tyr Tyr Thr Ser Asp Gly Thr Gln 3540 45 Gly Gln Thr Phe Trp Cys Asp His Ala Gly Ala Leu Leu Gly Asn Ser 5055 60 Tyr Val Asp Asn Thr Ser Lys Val Thr Ala Asn Phe Val Lys Glu Thr 6570 75 80 Ala Ser Pro Thr Ser Thr Tyr Asp Thr Tyr Val Glu Phe Gly Phe Ala85 90 95 Ser Gly Ala Ala Thr Leu Lys Lys Gly Gln Phe Ile Thr Ile Gln Gly100 105 110 Arg Ile Thr Lys Ser Asp Trp Ser Asn Tyr Thr Gln Thr Asn AspTyr 115 120 125 Ser Phe Asp Ala Ser Ser Ser Thr Pro Val Val Asn Pro LysVal Thr 130 135 140 Gly Tyr Ile Gly Gly Ala Lys Val Leu Gly Thr Ala ProGly Ser Ala 145 150 155 160 Gly Leu Val Pro Arg Gly Ser Thr Ala Ile GlyMet Lys Glu Thr Ala 165 170 175 Ala Ala Lys Phe Glu Arg Gln His Met AspSer Pro Asp Leu Gly Thr 180 185 190 Asp Asp Asp Asp Lys Met Gly Val LysVal Leu Phe Ala Leu Ile Cys 195 200 205 Ile Ala Val Ala Glu Ala Lys ProThr Glu Asn Asn Glu Asp Phe Asn 210 215 220 Ile Val Ala Val Ala Ser AsnPhe Ala Thr Thr Asp Leu Asp Ala Asp 225 230 235 240 Arg Gly Lys Leu ProGly Lys Lys Leu Pro Leu Glu Val Leu Lys Glu 245 250 255 Met Glu Ala AsnAla Arg Lys Ala Gly Cys Thr Arg Gly Cys Leu Ile 260 265 270 Cys Leu SerHis Ile Lys Cys Thr Pro Lys Met Lys Lys Phe Ile Pro 275 280 285 Gly ArgCys His Thr Tyr Glu Gly Asp Lys Glu Ser Ala Gln Gly Gly 290 295 300 IleGly Glu Ala Ile Val Asp Ile Pro Glu Ile Pro Gly Phe Lys Asp 305 310 315320 Leu Glu Pro Met Glu Gln Phe Ile Ala Gln Val Asp Leu Cys Val Asp 325330 335 Cys Thr Thr Gly Cys Leu Lys Gly Leu Ala Asn Val Gln Cys Ser Asp340 345 350 Leu Leu Lys Lys Trp Leu Pro Gln Arg Cys Ala Thr Phe Ala SerLys 355 360 365 Ile Gln Gly Gln Val Asp Lys Ile Lys Gly Ala Gly Gly Asp370 375 380 23 864 DNA Renilla renifomis CDS (61)...(762) GFP Clone-1 23ggcacgaggg tttcctgaca caataaaaac ctttcaaatt gtttctctgt agcagtaagt 60 atggat ctc gca aaa ctt ggt ttg aag gaa gtg atg cct act aaa atc 108 Met AspLeu Ala Lys Leu Gly Leu Lys Glu Val Met Pro Thr Lys Ile 1 5 10 15 aactta gaa gga ctg gtt ggc gac cac gct ttc tca atg gaa gga gtt 156 Asn LeuGlu Gly Leu Val Gly Asp His Ala Phe Ser Met Glu Gly Val 20 25 30 ggc gaaggc aac ata ttg gaa gga act caa gag gtg aag ata tcg gta 204 Gly Glu GlyAsn Ile Leu Glu Gly Thr Gln Glu Val Lys Ile Ser Val 35 40 45 aca aaa ggcgca cca ctc cca ttc gca ttt gat atc gta tct gtg gct 252 Thr Lys Gly AlaPro Leu Pro Phe Ala Phe Asp Ile Val Ser Val Ala 50 55 60 ttt tca tat gggaac aga gct tat acc ggt tac cca gaa gaa att tcc 300 Phe Ser Tyr Gly AsnArg Ala Tyr Thr Gly Tyr Pro Glu Glu Ile Ser 65 70 75 80 gac tac ttc ctccag tcg ttt cca gaa ggc ttt act tac gag aga aac 348 Asp Tyr Phe Leu GlnSer Phe Pro Glu Gly Phe Thr Tyr Glu Arg Asn 85 90 95 att cgt tat caa gatgga gga act gca att gtt aaa tct gat ata agc 396 Ile Arg Tyr Gln Asp GlyGly Thr Ala Ile Val Lys Ser Asp Ile Ser 100 105 110 ttg gaa gat ggt aaattc ata gtg aat gta gac ttc aaa gcg aag gat 444 Leu Glu Asp Gly Lys PheIle Val Asn Val Asp Phe Lys Ala Lys Asp 115 120 125 cta cgt cgc atg ggacca gtc atg cag caa gac atc gtg ggt atg cag 492 Leu Arg Arg Met Gly ProVal Met Gln Gln Asp Ile Val Gly Met Gln 130 135 140 cca tcg tat gag tcaatg tac acc aat gtc act tca gtt ata ggg gaa 540 Pro Ser Tyr Glu Ser MetTyr Thr Asn Val Thr Ser Val Ile Gly Glu 145 150 155 160 tgt ata ata gcattc aaa ctt caa act ggc aag cat ttc act tac cac 588 Cys Ile Ile Ala PheLys Leu Gln Thr Gly Lys His Phe Thr Tyr His 165 170 175 atg agg aca gtttac aaa tca aag aag cca gtg gaa act atg cca ttg 636 Met Arg Thr Val TyrLys Ser Lys Lys Pro Val Glu Thr Met Pro Leu 180 185 190 tat cat ttc atccag cat cgc ctc gtt aag acc aat gtg gac aca gcc 684 Tyr His Phe Ile GlnHis Arg Leu Val Lys Thr Asn Val Asp Thr Ala 195 200 205 agt ggt tac gttgtg caa cac gag aca gca att gca gcg cat tct aca 732 Ser Gly Tyr Val ValGln His Glu Thr Ala Ile Ala Ala His Ser Thr 210 215 220 atc aaa aaa attgaa ggc tct tta cca tag atacctgtac acaattattc 782 Ile Lys Lys Ile GluGly Ser Leu Pro * 225 230 tatgcacgta gcattttttt ggaaatataa gtggtattgttcaataaaat attaaatata 842 aaaaaaaaaa aaaaaaaaaa aa 864 24 860 DNARenilla renifromis CDS (57)...(758) GFP Clone-2 24 ggcacgaggc tgacacaataaaaaaccttt caaattgttt ctctgtagca ggaagt atg 59 Met 1 gat ctc gca aaa cttggt ttg aag gaa gtg atg cct act aaa atc aac 107 Asp Leu Ala Lys Leu GlyLeu Lys Glu Val Met Pro Thr Lys Ile Asn 5 10 15 tta gaa gga ctg gtt ggcgac cac gct ttc tca atg gaa gga gtt ggc 155 Leu Glu Gly Leu Val Gly AspHis Ala Phe Ser Met Glu Gly Val Gly 20 25 30 gaa ggc aac ata ttg gaa ggaact caa gag gtg aag ata tcg gta aca 203 Glu Gly Asn Ile Leu Glu Gly ThrGln Glu Val Lys Ile Ser Val Thr 35 40 45 aaa ggc gca cca ctc cca ttc gcattt gat atc gta tct gtt gct ttc 251 Lys Gly Ala Pro Leu Pro Phe Ala PheAsp Ile Val Ser Val Ala Phe 50 55 60 65 tca tat ggg aac aga gct tat actggt tac cca gaa gaa att tcc gac 299 Ser Tyr Gly Asn Arg Ala Tyr Thr GlyTyr Pro Glu Glu Ile Ser Asp 70 75 80 tac ttc ctc cag tcg ttt cca gaa ggcttt act tac gag aga aac att 347 Tyr Phe Leu Gln Ser Phe Pro Glu Gly PheThr Tyr Glu Arg Asn Ile 85 90 95 cgt tat caa gat gga gga act gca att gttaaa tct gat ata agc ttg 395 Arg Tyr Gln Asp Gly Gly Thr Ala Ile Val LysSer Asp Ile Ser Leu 100 105 110 gaa gat ggt aaa ttc ata gtg aat gta gacttc aaa gcg aag gat cta 443 Glu Asp Gly Lys Phe Ile Val Asn Val Asp PheLys Ala Lys Asp Leu 115 120 125 cgt cgc atg gga cca gtc atg cag caa gacatc gtg ggt atg cag cca 491 Arg Arg Met Gly Pro Val Met Gln Gln Asp IleVal Gly Met Gln Pro 130 135 140 145 tcg tat gag tca atg tac acc aat gtcact tca gtt ata ggg gaa tgt 539 Ser Tyr Glu Ser Met Tyr Thr Asn Val ThrSer Val Ile Gly Glu Cys 150 155 160 ata ata gca ttc aaa ctt caa act ggcaaa cat ttc act tac cac atg 587 Ile Ile Ala Phe Lys Leu Gln Thr Gly LysHis Phe Thr Tyr His Met 165 170 175 agg aca gtt tac aaa tca aag aag ccagtg gaa act atg cca ttg tat 635 Arg Thr Val Tyr Lys Ser Lys Lys Pro ValGlu Thr Met Pro Leu Tyr 180 185 190 cat ttc atc cag cat cgc ctc gtt aagacc aat gtg gac aca gcc agt 683 His Phe Ile Gln His Arg Leu Val Lys ThrAsn Val Asp Thr Ala Ser 195 200 205 ggt tac gtt gtg caa cac gag aca gcaatt gca gcg cat tct aca atc 731 Gly Tyr Val Val Gln His Glu Thr Ala IleAla Ala His Ser Thr Ile 210 215 220 225 aaa aaa att gaa ggc tct tta ccatag atatctatac acaattattc 778 Lys Lys Ile Glu Gly Ser Leu Pro * 230tatgcacgta gcattttttt ggaaatataa gtggtattgt tcaataaaat attaaatata 838aaaaaaaaaa aaaaaaaaaa aa 860 25 873 DNA Renilla reniformis CDS(61)...(762) GFP Clone-3 25 ggcacgaggg tttcctgaca caataaaaac ctttcaaattgtttctctgt agcagtaagt 60 atg gat ctc gca aaa ctt ggt ttg aag gaa gtg atgcct act aaa atc 108 Met Asp Leu Ala Lys Leu Gly Leu Lys Glu Val Met ProThr Lys Ile 1 5 10 15 aac tta gaa gga ctg gtt ggc gac cac gct ttc tcaatg gaa gga gtt 156 Asn Leu Glu Gly Leu Val Gly Asp His Ala Phe Ser MetGlu Gly Val 20 25 30 ggc gaa ggc aac ata ttg gaa gga act caa gag gtg aagata tcg gta 204 Gly Glu Gly Asn Ile Leu Glu Gly Thr Gln Glu Val Lys IleSer Val 35 40 45 aca aaa ggc gca cca ctc cca ttc gca ttt gat atc gta tctgtg gct 252 Thr Lys Gly Ala Pro Leu Pro Phe Ala Phe Asp Ile Val Ser ValAla 50 55 60 ttt tca tat ggg aac aga gct tat acc ggt tac cca gaa gaa atttcc 300 Phe Ser Tyr Gly Asn Arg Ala Tyr Thr Gly Tyr Pro Glu Glu Ile Ser65 70 75 80 gac tac ttc ctc cag tcg ttt cca gaa ggc ttt act tac gag agaaac 348 Asp Tyr Phe Leu Gln Ser Phe Pro Glu Gly Phe Thr Tyr Glu Arg Asn85 90 95 att cgt tat caa gat gga gga act gca att gtt aaa tct gat ata agc396 Ile Arg Tyr Gln Asp Gly Gly Thr Ala Ile Val Lys Ser Asp Ile Ser 100105 110 ttg gaa gat ggt aaa ttc ata gtg aat gta gac ttc aaa gcg aag gat444 Leu Glu Asp Gly Lys Phe Ile Val Asn Val Asp Phe Lys Ala Lys Asp 115120 125 cta cgt cgc atg gga cca gtc atg cag caa gac atc gtg ggt atg cag492 Leu Arg Arg Met Gly Pro Val Met Gln Gln Asp Ile Val Gly Met Gln 130135 140 cca tcg tat gag tca atg tac acc aat gtc act tca gtt ata ggg gaa540 Pro Ser Tyr Glu Ser Met Tyr Thr Asn Val Thr Ser Val Ile Gly Glu 145150 155 160 tgt ata ata gca ttc aaa ctt caa act ggc aag cat ttc act taccac 588 Cys Ile Ile Ala Phe Lys Leu Gln Thr Gly Lys His Phe Thr Tyr His165 170 175 atg agg aca gtt tac aaa tca aag aag cca gtg gaa act atg ccattg 636 Met Arg Thr Val Tyr Lys Ser Lys Lys Pro Val Glu Thr Met Pro Leu180 185 190 tat cat ttc atc cag cat cgc ctc gtt aag acc aat gtg gac acagcc 684 Tyr His Phe Ile Gln His Arg Leu Val Lys Thr Asn Val Asp Thr Ala195 200 205 agt ggt tac gtt gtg caa cac gag aca gca att gca gcg cat tctaca 732 Ser Gly Tyr Val Val Gln His Glu Thr Ala Ile Ala Ala His Ser Thr210 215 220 atc aaa aaa att gaa ggc tct tta cca tag atacctgtacacaattattc 782 Ile Lys Lys Ile Glu Gly Ser Leu Pro * 225 230 tatgcacgtagcattttttt ggaaatataa gtggtattgt tcaataaaat attaaatata 842 tgcttttgcaaaaaaaaaaa aaaaaaaaaa a 873 26 864 DNA Renilla reniformis CDS(61)...(759) Human codon optimized Renilla reniformis GFP 26 ggcacgagggtttcctgaca caataaaaac ctttcaaatt gtttctctgt agcagtaagt 60 atg gac ctggcc aag ctg ggc ctg aag gag gtg atg ccc acc aag atc 108 Met Asp Leu AlaLys Leu Gly Leu Lys Glu Val Met Pro Thr Lys Ile 1 5 10 15 aac ctg gagggc ctg gtg ggc gac cac gcc ttc tcg atg gag ggc gtg 156 Asn Leu Glu GlyLeu Val Gly Asp His Ala Phe Ser Met Glu Gly Val 20 25 30 ggc gag ggc aacatc ttg gag ggc acc cag gag gtg aag atc agc gtg 204 Gly Glu Gly Asn IleLeu Glu Gly Thr Gln Glu Val Lys Ile Ser Val 35 40 45 acc aag ggc gcc cccctg ccc ttc gcc ttc gac atc gtg agc gtg gcc 252 Thr Lys Gly Ala Pro LeuPro Phe Ala Phe Asp Ile Val Ser Val Ala 50 55 60 ttc agc tac ggc aac cgggcc tac acc ggc tac ccc gag gag atc agc 300 Phe Ser Tyr Gly Asn Arg AlaTyr Thr Gly Tyr Pro Glu Glu Ile Ser 65 70 75 80 gac tac ttc ctg cag agcttc ccc gag ggc ttc acc tac gag cgg aac 348 Asp Tyr Phe Leu Gln Ser PhePro Glu Gly Phe Thr Tyr Glu Arg Asn 85 90 95 atc cgg tac cag gac ggc ggcacc gcc atc gtg aag agc gac atc agc 396 Ile Arg Tyr Gln Asp Gly Gly ThrAla Ile Val Lys Ser Asp Ile Ser 100 105 110 ctg gag gac ggc aag ttc atcgtg aac gtg gac ttc aag gcc aag gac 444 Leu Glu Asp Gly Lys Phe Ile ValAsn Val Asp Phe Lys Ala Lys Asp 115 120 125 ctg cgg cgg atg ggc ccc gtgatg cag cag gac atc gtg ggc atg cag 492 Leu Arg Arg Met Gly Pro Val MetGln Gln Asp Ile Val Gly Met Gln 130 135 140 ccc agc tac gag agc atg tacacc aac gtg acc agc gtg atc ggc gag 540 Pro Ser Tyr Glu Ser Met Tyr ThrAsn Val Thr Ser Val Ile Gly Glu 145 150 155 160 tgc atc atc gcc ttc aagctg cag acc ggc aag cac ttc acc tac cac 588 Cys Ile Ile Ala Phe Lys LeuGln Thr Gly Lys His Phe Thr Tyr His 165 170 175 atg cgg acc gtg tac aagagc aag aag ccc gtg gag acc atg ccc ctg 636 Met Arg Thr Val Tyr Lys SerLys Lys Pro Val Glu Thr Met Pro Leu 180 185 190 tac cac ttc atc cag caccgg ctg gtg aag acc aac gtg gac acc gcc 684 Tyr His Phe Ile Gln His ArgLeu Val Lys Thr Asn Val Asp Thr Ala 195 200 205 agc ggc tac gtg gtg cagcac gag aca gcc atc gcc gcc cac agc acc 732 Ser Gly Tyr Val Val Gln HisGlu Thr Ala Ile Ala Ala His Ser Thr 210 215 220 atc aag aag atc gag ggcagc ctg ccc tagatacctg tacacaatta 779 Ile Lys Lys Ile Glu Gly Ser LeuPro 225 230 ttctatgcac gtagcatttt tttggaaata taagtggtat tgttcaataaaatattaaat 839 ataaaaaaaa aaaaaaaaaa aaaaa 864 27 233 PRT Renillareniformis 27 Met Asp Leu Ala Lys Leu Gly Leu Lys Glu Val Met Pro ThrLys Ile 1 5 10 15 Asn Leu Glu Gly Leu Val Gly Asp His Ala Phe Ser MetGlu Gly Val 20 25 30 Gly Glu Gly Asn Ile Leu Glu Gly Thr Gln Glu Val LysIle Ser Val 35 40 45 Thr Lys Gly Ala Pro Leu Pro Phe Ala Phe Asp Ile ValSer Val Ala 50 55 60 Phe Ser Tyr Gly Asn Arg Ala Tyr Thr Gly Tyr Pro GluGlu Ile Ser 65 70 75 80 Asp Tyr Phe Leu Gln Ser Phe Pro Glu Gly Phe ThrTyr Glu Arg Asn 85 90 95 Ile Arg Tyr Gln Asp Gly Gly Thr Ala Ile Val LysSer Asp Ile Ser 100 105 110 Leu Glu Asp Gly Lys Phe Ile Val Asn Val AspPhe Lys Ala Lys Asp 115 120 125 Leu Arg Arg Met Gly Pro Val Met Gln GlnAsp Ile Val Gly Met Gln 130 135 140 Pro Ser Tyr Glu Ser Met Tyr Thr AsnVal Thr Ser Val Ile Gly Glu 145 150 155 160 Cys Ile Ile Ala Phe Lys LeuGln Thr Gly Lys His Phe Thr Tyr His 165 170 175 Met Arg Thr Val Tyr LysSer Lys Lys Pro Val Glu Thr Met Pro Leu 180 185 190 Tyr His Phe Ile GlnHis Arg Leu Val Lys Thr Asn Val Asp Thr Ala 195 200 205 Ser Gly Tyr ValVal Gln His Glu Thr Ala Ile Ala Ala His Ser Thr 210 215 220 Ile Lys LysIle Glu Gly Ser Leu Pro 225 230 28 861 DNA Pleuromamma CDS (148)...(741)Pleuormamma luciferase 28 cggcacgaga ttttgtctgt ggtgattggg attgtctgtctctcaggtca agcagaaagt 60 tcgctgaaag gtgatttctg tagtgatgtt tccttctgggatgtgatcaa gtacaacact 120 gagagtcgac aatgctgtga cacaaaa atg ctt aga aattgc gct agg aag caa 174 Met Leu Arg Asn Cys Ala Arg Lys Gln 1 5 gag caagtt tgc gcc gat gtg acc gag atg aaa tgc caa gca gtt gct 222 Glu Gln ValCys Ala Asp Val Thr Glu Met Lys Cys Gln Ala Val Ala 10 15 20 25 tgg gccgac tgt gga ccc aga ttt gat tcc act ggc agg aat aga tgc 270 Trp Ala AspCys Gly Pro Arg Phe Asp Ser Thr Gly Arg Asn Arg Cys 30 35 40 caa gtt caatac aag gac tac gcg tac aag tcc tgc gtg gaa gtt gat 318 Gln Val Gln TyrLys Asp Tyr Ala Tyr Lys Ser Cys Val Glu Val Asp 45 50 55 tac act gta ccgcac agg aag caa gtt cca gag tgc aaa caa gtc act 366 Tyr Thr Val Pro HisArg Lys Gln Val Pro Glu Cys Lys Gln Val Thr 60 65 70 aaa gat aac tgc gttact gat tgg gaa gtt gac gcc aat ggc aac aag 414 Lys Asp Asn Cys Val ThrAsp Trp Glu Val Asp Ala Asn Gly Asn Lys 75 80 85 gtt tgg ggt ggt acc gagaaa tgc act cct gtc act tgg gaa gaa tgt 462 Val Trp Gly Gly Thr Glu LysCys Thr Pro Val Thr Trp Glu Glu Cys 90 95 100 105 aat atc gtg gag aaagat gta gat ttt cca act gtc aag acg gaa tgc 510 Asn Ile Val Glu Lys AspVal Asp Phe Pro Thr Val Lys Thr Glu Cys 110 115 120 ggc atc ctg tct cacctt aag tat gca gac ttc ata gag gga cct tcc 558 Gly Ile Leu Ser His LeuLys Tyr Ala Asp Phe Ile Glu Gly Pro Ser 125 130 135 cac tct ttg tct atgaga acc aat tgt cag gtc aag agt tca ttg gac 606 His Ser Leu Ser Met ArgThr Asn Cys Gln Val Lys Ser Ser Leu Asp 140 145 150 tgc cgg cct gtt aagacc agg aag tgt gca acg gtc gag tac cac gaa 654 Cys Arg Pro Val Lys ThrArg Lys Cys Ala Thr Val Glu Tyr His Glu 155 160 165 tgc agc atg aag ccccaa gaa gac tgc agc cca gtc act gtt cat att 702 Cys Ser Met Lys Pro GlnGlu Asp Cys Ser Pro Val Thr Val His Ile 170 175 180 185 cct gac cag gagaaa gtt cac cag aag aag tgc ctc aca taaatgttat 751 Pro Asp Gln Glu LysVal His Gln Lys Lys Cys Leu Thr 190 195 caattttagc tcttactaat ttaaacataataaatatcac atcgaagccc tttattttat 811 agaagtgtaa tgcttgaata aatctagtgaataaaaaaaa aaaaaaaaaa 861 29 198 PRT Pleuromamma 29 Met Leu Arg Asn CysAla Arg Lys Gln Glu Gln Val Cys Ala Asp Val 1 5 10 15 Thr Glu Met LysCys Gln Ala Val Ala Trp Ala Asp Cys Gly Pro Arg 20 25 30 Phe Asp Ser ThrGly Arg Asn Arg Cys Gln Val Gln Tyr Lys Asp Tyr 35 40 45 Ala Tyr Lys SerCys Val Glu Val Asp Tyr Thr Val Pro His Arg Lys 50 55 60 Gln Val Pro GluCys Lys Gln Val Thr Lys Asp Asn Cys Val Thr Asp 65 70 75 80 Trp Glu ValAsp Ala Asn Gly Asn Lys Val Trp Gly Gly Thr Glu Lys 85 90 95 Cys Thr ProVal Thr Trp Glu Glu Cys Asn Ile Val Glu Lys Asp Val 100 105 110 Asp PhePro Thr Val Lys Thr Glu Cys Gly Ile Leu Ser His Leu Lys 115 120 125 TyrAla Asp Phe Ile Glu Gly Pro Ser His Ser Leu Ser Met Arg Thr 130 135 140Asn Cys Gln Val Lys Ser Ser Leu Asp Cys Arg Pro Val Lys Thr Arg 145 150155 160 Lys Cys Ala Thr Val Glu Tyr His Glu Cys Ser Met Lys Pro Gln Glu165 170 175 Asp Cys Ser Pro Val Thr Val His Ile Pro Asp Gln Glu Lys ValHis 180 185 190 Gln Lys Lys Cys Leu Thr 195 30 1104 DNA Ptilosarcusgurneyi CDS (34)...(747) Ptilosarcus Green Flourescent Protein 30tcggcacgag ctggcctcca cactttagac aaa atg aac cgc aac gta tta aag 54 MetAsn Arg Asn Val Leu Lys 1 5 aac act gga ctg aaa gag att atg tcg gca aaagct agc gtt gaa gga 102 Asn Thr Gly Leu Lys Glu Ile Met Ser Ala Lys AlaSer Val Glu Gly 10 15 20 atc gtg aac aat cac gtt ttt tcc atg gaa gga tttgga aaa ggc aat 150 Ile Val Asn Asn His Val Phe Ser Met Glu Gly Phe GlyLys Gly Asn 25 30 35 gta tta ttt gga aac caa ttg atg caa atc cgg gtt acaaag gga ggt 198 Val Leu Phe Gly Asn Gln Leu Met Gln Ile Arg Val Thr LysGly Gly 40 45 50 55 ccg ttg cca ttc gct ttc gat att gtt tcc ata gct ttccaa tac ggg 246 Pro Leu Pro Phe Ala Phe Asp Ile Val Ser Ile Ala Phe GlnTyr Gly 60 65 70 aat cgc act ttc acg aaa tac cca gac gac att gcg gac tacttt gtt 294 Asn Arg Thr Phe Thr Lys Tyr Pro Asp Asp Ile Ala Asp Tyr PheVal 75 80 85 caa tca ttc ccg gct gga ttt ttc tac gaa aga aat cta cgc tttgaa 342 Gln Ser Phe Pro Ala Gly Phe Phe Tyr Glu Arg Asn Leu Arg Phe Glu90 95 100 gat ggc gcc att gtt gac att cgt tca gat ata agt tta gaa gatgat 390 Asp Gly Ala Ile Val Asp Ile Arg Ser Asp Ile Ser Leu Glu Asp Asp105 110 115 aag ttc cac tac aaa gtg gag tat aga ggc aac ggt ttc cct agtaac 438 Lys Phe His Tyr Lys Val Glu Tyr Arg Gly Asn Gly Phe Pro Ser Asn120 125 130 135 gga ccc gtg atg caa aaa gcc atc ctc ggc atg gag cca tcgttt gag 486 Gly Pro Val Met Gln Lys Ala Ile Leu Gly Met Glu Pro Ser PheGlu 140 145 150 gtg gtc tac atg aac agc ggc gtt ctg gtg ggc gaa gta gatctc gtt 534 Val Val Tyr Met Asn Ser Gly Val Leu Val Gly Glu Val Asp LeuVal 155 160 165 tac aaa ctc gag tca ggg aac tat tac tcg tgc cac atg aaaacg ttt 582 Tyr Lys Leu Glu Ser Gly Asn Tyr Tyr Ser Cys His Met Lys ThrPhe 170 175 180 tac aga tcc aaa ggt gga gtg aaa gaa ttc ccg gaa tat cacttt atc 630 Tyr Arg Ser Lys Gly Gly Val Lys Glu Phe Pro Glu Tyr His PheIle 185 190 195 cat cat cgt ctg gag aaa acc tac gtg gaa gaa gga agc ttcgtg gaa 678 His His Arg Leu Glu Lys Thr Tyr Val Glu Glu Gly Ser Phe ValGlu 200 205 210 215 caa cac gag acg gcc att gca caa ctg acc aca att ggaaaa cct ctg 726 Gln His Glu Thr Ala Ile Ala Gln Leu Thr Thr Ile Gly LysPro Leu 220 225 230 ggc tcc ctt cat gaa tgg gtg tagaaaatga ccaatatactggggaaaccg 777 Gly Ser Leu His Glu Trp Val 235 ataaccgttt ggaagcttgtgtatacaaat tatttggggt cattttgtaa tgtgtatgtg 837 tgttgtatga tcaatagacgtcgtcattca tagcttgaat ccttcagcaa aagaaacctc 897 gaagcatatt gaaacctcgaagcatattga aacctcgacg gagagcgtaa agagaccgca 957 caaattaacg cgtttcaaccagcagttgga atctttaaac cgatcaaaac tattaatata 1017 aatatatata ccctgtataacttatatata tctatatagt ttgatattga ttaaatctgt 1077 tcttgatcaa aaaaaaaaaaaaaaaaa 1104 31 1279 DNA Ptilosarcus gurneyi CDS (7)...(720) PtilosarcusGreen Flourescent Protein (GFP) 31 gacaaa atg aac cgc aac gta tta aagaac act gga ctg aaa gag att 48 Met Asn Arg Asn Val Leu Lys Asn Thr GlyLeu Lys Glu Ile 1 5 10 atg tcg gca aaa gct agc gtt gaa gga atc gtg aacaat cac gtt ttt 96 Met Ser Ala Lys Ala Ser Val Glu Gly Ile Val Asn AsnHis Val Phe 15 20 25 30 tcc atg gaa gga ttt gga aaa ggc aat gta tta tttgga aac caa ttg 144 Ser Met Glu Gly Phe Gly Lys Gly Asn Val Leu Phe GlyAsn Gln Leu 35 40 45 atg caa atc cgg gtt aca aag gga ggt ccg ttg cca ttcgct ttc gac 192 Met Gln Ile Arg Val Thr Lys Gly Gly Pro Leu Pro Phe AlaPhe Asp 50 55 60 att gtt tcc ata gct ttc caa tac ggg aat cgc act ttc acgaaa tac 240 Ile Val Ser Ile Ala Phe Gln Tyr Gly Asn Arg Thr Phe Thr LysTyr 65 70 75 cca gac gac att gcg gac tac ttt gtt caa tca ttt ccg gct ggattt 288 Pro Asp Asp Ile Ala Asp Tyr Phe Val Gln Ser Phe Pro Ala Gly Phe80 85 90 ttc tac gaa aga aat cta cgc ttt gaa gat ggc gcc att gtt gac att336 Phe Tyr Glu Arg Asn Leu Arg Phe Glu Asp Gly Ala Ile Val Asp Ile 95100 105 110 cgt tca gat ata agt tta gaa gat gat aag ttc cac tac aaa gtggag 384 Arg Ser Asp Ile Ser Leu Glu Asp Asp Lys Phe His Tyr Lys Val Glu115 120 125 tat aga ggc aac ggt ttc cct agt aac gga ccc gtg atg caa aaagcc 432 Tyr Arg Gly Asn Gly Phe Pro Ser Asn Gly Pro Val Met Gln Lys Ala130 135 140 atc ctc ggc atg gag cca tcg ttt gag gtg gtc tac atg aac agcggc 480 Ile Leu Gly Met Glu Pro Ser Phe Glu Val Val Tyr Met Asn Ser Gly145 150 155 gtt ctg gtg ggc gaa gta gat ctc gtt tac aaa ctc gag tca gggaac 528 Val Leu Val Gly Glu Val Asp Leu Val Tyr Lys Leu Glu Ser Gly Asn160 165 170 tat tac tcg tgc cac atg aaa acg ttt tac aga tcc aaa ggt ggagtg 576 Tyr Tyr Ser Cys His Met Lys Thr Phe Tyr Arg Ser Lys Gly Gly Val175 180 185 190 aaa gaa ttc ccg gaa tat cac ttt atc cat cat cgt ctg gagaaa acc 624 Lys Glu Phe Pro Glu Tyr His Phe Ile His His Arg Leu Glu LysThr 195 200 205 tac gtg gaa gaa gga agc ttc gtg gaa caa cac gag acg gccatt gca 672 Tyr Val Glu Glu Gly Ser Phe Val Glu Gln His Glu Thr Ala IleAla 210 215 220 caa ctg acc aca att gga aaa cct ctg ggc tcc ctt cat gaatgg gtg 720 Gln Leu Thr Thr Ile Gly Lys Pro Leu Gly Ser Leu His Glu TrpVal 225 230 235 tagaaaatga ccaatatact ggggaaaatc accaatatac tggggaaaatgaccaattta 780 ctggggaaaa tgaccaatat actgtagaaa atcaccaata tactggggaaaatgaccaat 840 ttactgggga aatgaccaat ttactgtaga aaatcaccaa tatactgtggaaaatgacca 900 aaatactgta gaaatgttca cactgggttg ataaccgttt cgataaccgtttggaagctt 960 gtgtatacaa gttatttggg gtcattttgt aatgtgtatg tgtgttgtatgatctataga 1020 cgtcgtcatt catagcttga atccttcagc aaaagaaacc tcgaagcatattgaaacctc 1080 gacggagagc ataaagagac cgcacgtaca caaattataa taccagcagttggaatcttt 1140 aaaccgatca aaactattaa tatatatata caccctgtat aacatatatatatatatata 1200 tctacatagt ttgatattga ttaaatctgt tcttgatcac taaaaaaaaaaaaaaaaaaa 1260 aaaaaaaaaa aaaaaaaaa 1279 32 238 PRT Ptilosarcus gurneyi32 Met Asn Arg Asn Val Leu Lys Asn Thr Gly Leu Lys Glu Ile Met Ser 1 510 15 Ala Lys Ala Ser Val Glu Gly Ile Val Asn Asn His Val Phe Ser Met 2025 30 Glu Gly Phe Gly Lys Gly Asn Val Leu Phe Gly Asn Gln Leu Met Gln 3540 45 Ile Arg Val Thr Lys Gly Gly Pro Leu Pro Phe Ala Phe Asp Ile Val 5055 60 Ser Ile Ala Phe Gln Tyr Gly Asn Arg Thr Phe Thr Lys Tyr Pro Asp 6570 75 80 Asp Ile Ala Asp Tyr Phe Val Gln Ser Phe Pro Ala Gly Phe Phe Tyr85 90 95 Glu Arg Asn Leu Arg Phe Glu Asp Gly Ala Ile Val Asp Ile Arg Ser100 105 110 Asp Ile Ser Leu Glu Asp Asp Lys Phe His Tyr Lys Val Glu TyrArg 115 120 125 Gly Asn Gly Phe Pro Ser Asn Gly Pro Val Met Gln Lys AlaIle Leu 130 135 140 Gly Met Glu Pro Ser Phe Glu Val Val Tyr Met Asn SerGly Val Leu 145 150 155 160 Val Gly Glu Val Asp Leu Val Tyr Lys Leu GluSer Gly Asn Tyr Tyr 165 170 175 Ser Cys His Met Lys Thr Phe Tyr Arg SerLys Gly Gly Val Lys Glu 180 185 190 Phe Pro Glu Tyr His Phe Ile His HisArg Leu Glu Lys Thr Tyr Val 195 200 205 Glu Glu Gly Ser Phe Val Glu GlnHis Glu Thr Ala Ile Ala Gln Leu 210 215 220 Thr Thr Ile Gly Lys Pro LeuGly Ser Leu His Glu Trp Val 225 230 235 33 233 PRT Renilla Reniformismutein 33 Met Asp Leu Ala Lys Leu Gly Leu Lys Glu Val Met Pro Thr LysIle 1 5 10 15 Asn Leu Glu Gly Leu Val Gly Asp His Ala Phe Ser Met GluGly Val 20 25 30 Gly Glu Gly Asn Ile Leu Glu Gly Thr Gln Glu Val Lys IleSer Val 35 40 45 Thr Lys Gly Ala Pro Leu Pro Phe Ala Phe Asp Ile Val SerVal Ala 50 55 60 Phe Ser Tyr Gly Asn Arg Ala Tyr Thr Gly Tyr Pro Glu GluIle Ser 65 70 75 80 Asp Tyr Phe Leu Gln Ser Phe Pro Glu Gly Phe Thr TyrGlu Arg Asn 85 90 95 Ile Arg Tyr Gln Asp Gly Gly Thr Ala Ile Val Asp SerAsp Ile Ser 100 105 110 Leu Glu Asp Gly Lys Phe Ile Val Asn Val Asp PheLys Ala Asp Asp 115 120 125 Leu Arg Asp Met Gly Pro Val Met Gln Gln AspIle Val Gly Met Gln 130 135 140 Pro Ser Tyr Glu Ser Met Tyr Thr Asn ValThr Ser Val Ile Gly Glu 145 150 155 160 Cys Ile Ile Ala Phe Lys Leu GlnThr Gly Lys Asp Phe Thr Tyr His 165 170 175 Met Arg Thr Val Tyr Lys SerLys Lys Pro Val Glu Thr Met Pro Leu 180 185 190 Tyr His Phe Ile Gln HisAsp Leu Val Lys Thr Asn Val Asp Thr Ala 195 200 205 Ser Gly Tyr Val ValGln His Glu Thr Ala Ile Ala Ala His Ser Thr 210 215 220 Ile Asp Lys IleGlu Gly Ser Leu Pro 225 230

What is claimed is:
 1. An isolated nucleic acid molecule encoding aRenilla reniformis green fluorescent protein, comprising a sequence ofnucleotides that encodes the protein of SEQ ID No. 27 or a greenfluorescent protein encoded by a Renilla reniformis having at least 80%sequence identity thereto.
 2. An isolated nucleic acid molecule of claim1 that encodes a protein having at least 90% sequence identity to theprotein of SEQ ID No.
 27. 3. The isolated nucleic acid molecule of claim1, comprising a sequence of nucleotides selected from the groupconsisting of: (a) the coding portion of the sequence of nucleotides setforth in any of SEQ ID Nos. 23-25; (b) a sequence of nucleotides thathybridizes under high stringency to the sequence of nucleotides of (a);and (c) a sequence of nucleotides comprising degenerate codons of (a) or(b).
 4. The isolated nucleic acid molecule of claim 1, wherein thenucleic acid is DNA.
 5. The isolated nucleic acid molecule of claim 1,wherein the nucleic acid is RNA.
 6. A nucleic acid probe or primer,comprising at least 14 contiguous nucleotides selected from the sequenceof nucleotides set of claim
 1. 7. The probe or primer of claim 6,comprising at least 16 contiguous nucleotides selected from the sequenceof nucleotides in claim
 1. 8. The probe or primer of claim 7, comprisingat least 30 contiguous nucleotides.
 9. A plasmid, comprising thesequence of nucleotides of claim
 1. 10. The plasmid of claim 8 that isan expression vector, comprising: a promoter element; a cloning site forthe introduction of nucleic acid; and a selectable marker; wherein thenucleic acid encoding the cloning site is positioned between nucleicacids encoding the promoter element and the green fluorescent proteinand wherein the nucleic acid encoding the green fluorescent protein isoperatively linked to the promoter element.
 11. The plasmid of claim 9,further comprising a sequence of nucleotides encoding a luciferase. 12.A recombinant host cell, comprising the plasmid of claim
 9. 13. The cellof claim 12, wherein the cell is selected from the group consisting of abacterial cell, a yeast cell, a fungal cell, a plant cell, an insectcell and an animal cell.
 14. An isolated substantially purified Renillareniformis green fluorescent protein (GFP) encoded by the nucleic acidmolecule of claim
 1. 15. A mutein of the GFP of claim 14 that exhibitsaltered spectral properties.
 16. A mutein of the GFP of claim 14 thatexhibits a reduced tendency to form multimers.
 17. A composition,comprising the green fluorescent protein of claim 14 and at least onecomponent of a bioluminescence generating system.
 18. The composition ofclaim 17, wherein the bioluminescence generating system is selected fromthose isolated from: an insect system, a coelenterate system, actenophore system, a bacterial system, a mollusk system, a crustaceasystem, a fish system, an annelid system, and an earthworm system. 19.The composition of claim 17, wherein the bioluminescence generatingsystem is selected from those isolated from: fireflies, Mnemiopsis,Beroe ovata, Aequorea, Obelia, Vargula, Pelagia, Renilla, PholasAristostomias, Pachystomias, Poricthys, Cypridina, Aristostomias, suchPachystomias, Malacosteus, Gonadostomias, Gaussia, Watensia, Halisturia,Vampire squid, Glyphus, Mycotophids, Vinciguerria, Howella,Florenciella, Chaudiodus, Melanocostus, Sea Pens, Chiroteuthis,Eucleoteuthis, Onychoteuthis, Watasenia, cuttlefish, Sepiolina,Oplophorus, Acanthophyra, Sergestes, Gnathophausia, Argyropelecus,Yarella, Diaphus, Gonadostomias and Neoscopelus.
 20. A mutein of claim15, comprising substitution in amino acids at amino acids 56-75 of SEQID No. 27, whereby spectral properties are altered.
 21. The compositionof claim 20, wherein the bioluminescence generating system is selectedfrom those isolated from Aequorea, Obelia, Vargula and Renilla.
 22. Areporter gene construct, comprising the nucleic acid of claim
 1. 23. Acombination, comprising: an article of manufacture; and a Renillareniformis green fluorescent protein (GFP) encoded by a nucleic acidmolecule of claim
 1. 24. The combination of claim 23, further comprisingat least one component of a bioluminescence generating system, wherebythe combination is a novelty item.
 25. The combination of claim 24,wherein the component of the bioluminescence generating system comprisesa luciferase.
 26. The combination of claim 24, wherein the component ofthe bioluminescence generating system comprises a comprises a luciferin.27. The combination of claim 23, wherein the article of manufacture isselected from among toys, fountains, personal care items, fairy dust,foods, textile and paper products.
 28. The combination of claim 27,wherein the article of manufacture is selected from among toy guns,pellet guns, greeting cards, fingerpaints, foot bags, slimy playmaterial, clothing, bubble making toys and bubbles therefor, balloons,bath powders, body lotions, gels, body powders, body creams,toothpastes, mouthwashes, soaps, body paints, bubble bath, board gametoys, fishing lures, egg-shaped toys, toy cigarettes, dolls, sparklers,magic wand toys, wrapping paper, gelatins, icings, frostings, fairydust, beer, ornamental transgenic plants, wine, champagne, milk, softdrinks, ice cubes, ice, dry ice, soaps, body paints and bubble bath. 29.The combination of claim 28 that is a transgenic ornamental plant. 30.The combination of claim 28 that is a toy.
 31. The combination of claim28 that is a food.
 32. The combination of claim 28 that is a cosmetic.33. The combination of claim 28 that is a beverage.
 34. The combinationof claim 24 wherein the article of manufacture is selected from amongtoys, fountains, personal care items, fairy dust, foods, textile,transgenic ornamental plant and paper products.
 35. The combination ofclaim 34, wherein the article of manufacture is selected from among toyguns, pellet guns, greeting cards, fingerpaints, foot bags, slimy playmaterial, clothing, bubble making toys and bubbles therefor, balloons,bath powders, body lotions, gels, body powders, body creams,toothpastes, mouthwashes, soaps, body paints, bubble bath, board gametoys, fishing lures, egg-shaped toys, toy cigarettes, dolls, sparklers,magic wand toys, wrapping paper, gelatins, icings, frostings, fairydust, beer, wine, champagne, soft drinks, ice cubes, ice, dry ice,soaps, body paints and bubble bath.
 36. An antibody that specificallybinds to Renilla reniformis or a molecule or derivative of the antibodycontaining the binding domain thereof. 37 The antibody of claim 36 thatis a monoclonal antibody.
 38. A nucleic acid construct, comprising anucleotide sequence encoding a luciferase and a sequence of nucleotidesof claim 1 that encodes a Renilla reniformis fluorescent protein (GFP).39. The nucleic acid construct of claim 38, wherein the luciferase is aRenilla mulleri luciferase, a Gaussia luciferase or a Pleuromammaluciferase.
 40. The nucleic acid construct of claim 39, wherein theGaussia luciferase is a Gaussia princepes luciferase.
 41. The nucleicacid construct of claim 38, wherein the luciferase is encoded by: asequence of nucleotides set forth in SEQ ID No. 17, SEQ ID No. 19, orSEQ ID No. 28; a sequence of nucleotides encoding the amino acidsequence set forth in SEQ ID No. 18, SEQ ID No. 20 or SEQ ID No. 29; anda sequence of nucleotides that hybridizes under high stringency to thesequence of nucleotides set forth in SEQ ID No. 17, SEQ ID No. 19 or SEQID No.
 28. 42. The nucleic acid construct of claim 38 that is DNA. 43.The nucleic acid construct of claim 38 that is RNA.
 44. A plasmid,comprising the nucleic acid construct of claim
 38. 45. The plasmid ofclaim 44, further comprising a sequence of nucleotides encoding: apromoter element; a selectable marker; wherein, the sequence ofnucleotides encoding the luciferase and GFP is operatively linked to thepromoter element, whereby the luciferase and GFP are expressed.
 46. Theconstruct of claim 38, wherein the luciferase and the GFP are encoded bya polycistronic message.
 47. The construct of claim 38, wherein theencoded luciferase and comprise a fusion protein.
 48. The construct ofclaim 38, wherein the luciferase is Renilla reniformis luciferase.
 49. Arecombinant host cell, comprising the plasmid of claim
 44. 50. The cellof claim 46, wherein the cell is selected from the group consisting of abacterial cell, a yeast cell, a fungal cell, a plant cell, an insectcell and an animal cell.
 51. An isolated substantially purifiedluciferase and GFP fusion protein, wherein the GFP is a Renillareniformis GFP and the fusion protein is encoded by the nucleic acidconstruct of claim
 47. 52. The fusion protein of claim 51, wherein theluciferase is a Renilla luciferase.
 53. The fusion protein of claim 51,wherein the luciferase and is a Renilla reniformis luciferase.
 54. Acomposition, comprising the fusion protein of claim
 48. 55. Thecomposition of claim 54, further comprising at least one component of abioluminescence generating system.
 56. The composition of claim 55,wherein the component of the bioluminescence generating system is aluciferin.
 57. The nucleic acid construct of claim 47, wherein thesequence of nucleotides encoding the luciferase and GFP are notcontiguous.
 58. The nucleic acid construct of claim 54, comprising asequence of nucleotides that encodes a ligand binding domain of a targetprotein.
 59. A biosensor, comprising a GFP protein encoded by thenucleic acid molecule of claim 1 and a luciferase.
 60. The biosensor ofclaim 59, wherein the luciferase is a Renilla luciferase.
 61. Abiosensor of claim 59, further comprising a modulator.
 62. A biosensor,comprising a fusion protein of claim
 51. 63. The biosensor of claim 62,wherein the GFP and luciferase in the fusion protein are not contiguous.64. A bioluminescence resonance energy transfer (BRET) system,comprising: (a) a GFP encoded by the nucleic molecule of claim 1; (b) aluciferase from which the GFP can accept energy when the GFP andluciferase; (c) a luciferin or other substrate of the luciferase. 65.The BRET system of claim 64, further comprising one or more modulators.66. The BRET system of claim 65, wherein the GFP and luciferase are eachattached to a different modulator, or each are attached to the samemodulator.
 67. The BRET system of claim 65, wherein a conformationchange in a modulator causes an increase in the proximity of theluciferase and GFP.
 68. The BRET system of claim 65, wherein aconformational change in a modulator causes a decrease in the proximityof the luciferase and GFP.
 69. The BRET system of claim 65, wherein theluciferase is Renilla reniformis luciferase.
 70. A microelectronicdevice, comprising: a substrate; a plurality of micro-locations definedon the substrate, wherein each micro-location is for linking amacromolecule; an independent photodetector integrated at or adjacent toeach micro-location and optically coupled to each micro-location, eachphotodetector being configured to generate a sensed signal responsive tothe photons of light emitted at the corresponding micro-location when alight-emitting chemical reaction occurs at that micro-location, eachphotodetector being independent from the photodetectors opticallycoupled to the other micro-locations; and an electronic circuit coupledto each photodetector and configured to read the sensed signal generatedby each photodetector and to generate output data signals therefrom thatare indicative of the light emitted at each micro-location by thelight-emitting chemical reactions, whereby the device detects photons oflight emitted by light-emitting chemical reactions, wherein: eachmicro-location is defined by a portion of the surface; themicro-locations defined on the substrate each comprise a components of abioluminescence generating system and a green fluorescent protein ofclaim 1, whereby photons of light are emitted when a reaction takesplace at that micro-location
 71. The device of claim 70, wherein themicro-locations are provided as an array.
 72. The device of claim 70,wherein the bioluminescence generating system comprises a Renillaluciferase.
 73. The device of claim 71, wherein the bioluminescencegenerating system comprises s Renilla reniformis luciferase.
 74. Amethod of detecting and identifying analytes in a biological sample,comprising: providing the microelectronic device of claim 70; attachinga macromolecule or plurality of different macromolecules to the surfaceat each micro-location on the device, wherein macromolecule is specificfor binding to selected analyte that may be present in the biologicalsample; contacting the sample with the surface of the microelectronicdevice, whereby any of the selected analytes that are present in thesample bind to the macromolecule attached to the surface at eachmicro-location; exposing the surface of the microelectronic device to asecond macromolecule or plurality thereof bind to the selected analytealready bound to the first macromolecule at each micro-location, whereinthe second macromolecule comprises a component of a bioluminescencegenerating reaction; initiating the bioluminescence generating reactionby contacting the surface of the device with the remaining components ofthe bioluminescence generating reaction, wherein the wavelength of theresulting light is shifted by the Renilla reniformis GFP; detectingphotons of light emitted by the GFP using a photodetector opticallycoupled to each micro-location, each photodetector generating a sensedsignal representative of the bioluminescence generation at therespective micro-location.
 75. A transgenic animal or plant thatexpresses the Renilla reniformis nucleic acid of claim
 1. 76. Thetransgenic animal or plant of claim 75, selected from among fish, worms,monkeys, rodents, goats, pigs, cows, sheep, horses, flowering plants,ornamental plants.
 77. The transgenic animal or plant of claim 75 thatis an orchid.